Enhanced immune cell receptor sequencing methods

ABSTRACT

Disclosed are methods for sequencing immune cell receptor repertoires from immune cell populations, and kits containing primer mixtures for the sequencing of immune cell receptor repertoires.

RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application Ser. No. 62/452,409, filed Jan. 31, 2017, the entire contents of which are incorporated herein by reference.

FIELD

The disclosure relates to methods, systems and kits for sequencing immune cell receptor repertoires from immune cells, such as T-cells or B-cells.

BACKGROUND

Immune cell repertoires, such as B- or T-cell repertoires, consists of millions of lymphocytes, each expressing a different protein complex that enables specific recognition of a single antigen. CD4 and CD8 positive T-cells express so-called T-cell receptors (TCRs). These heterodimeric receptors recognize antigen-derived peptides displayed by major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells, as described in Rudolph M G, Stanfield R L, Wilson I A. How TCRs bind MHCs, peptides, and coreceptors. Annu Rev Immunol. 2006; 24:419-66. TCRs are composed of two subunits, most commonly of one α and one β chain. A less common type of TCR contains one γ and one δ chain.

Alpha (a) chains consists of a (variable) V, a joining (J) and a constant (C) region, while beta (β) chains contain an additional diversity (D) region between the V and the J region (see FIG. 1), as described in Starr T K, Jameson S C, Hogquist K A. Positive and negative selection of T cells. Annu Rev Immunol. 2003; 21:139-76. Each of these TCR regions is encoded in several pieces, so-called gene segments, which are spatially segregated in the germline. In humans, the TCR α gene locus contains 54 different V gene segments, and 61 J gene segments. The human TCR β chain locus comprises 65 V, 2 D and 14 J segments. The great structural diversity of TCRs is achieved by somatic recombination of these TCR gene segments during lymphocyte development in the thymus. During this process, several gene segments of each region type are randomly selected and joined to form a rearranged TCR locus. Additional junctional diversity is created by the addition or removal of nucleotides at the sites of recombination, as described in Krangel M S. Mechanics of T cell receptor gene rearrangement. Curr Opin Immunol. 2009 April; 21(2):133-9. The process of V(D)J joining plays a critical role in shaping the third hypervariable loops (also called complementary determining regions, CDR3s) of the TCR α and β chains. These regions bind antigens and are essential for providing the high specificity of antigen recognition that TCRs exhibit.

Similarly to the TCR αβ, TCR gamma (γ) and delta (δ) segments undergo V(D)J rearrangement during thymus development. Both loci are recombined in the double negative (DN) stage of T-cell development. Differentiation towards γδ or αβ lineage relies on the ability of the cell to produce functional γδ or αβ TCR. The δ locus is embedded within the α locus. Dδ, Jδ and Cδ segments are located in between the V and the J segment of the α locus. The Vδ segments are the same as the Vα segments but only a fraction of the Vα segments are used for the TCR δ chain.

Overall, V(D)J recombination is able to generate millions of different TCR sequences and plays a critical role in an organism's ability to eliminate infections or transformed cells. Not surprisingly, TCR repertoires affect a wide range of diseases, including malignancy, autoimmune disorders and infectious diseases. TCR sequencing has been instrumental for our understanding of how the TCR repertoire evolves during infection or following treatment (e.g. after hematopoietic stem cell transplantation, chronical viral infection, immunotherapy). Further, the identification of TCRs on tumor-infiltrating lymphocytes and other T-cells that target cancer-specific epitopes has not only furthered our knowledge of malignant disease, but has also led to novel therapies for cancer such as adoptive T-cell transfer or cancer vaccines.

Due to the large diversity of sequences, determining TCR repertoires has been challenging in praxis. In the last couple of years, next generation sequencing (NGS) has opened up new opportunities to comprehensively assess the extreme diversity of TCR repertoires, as described in Genolet R, Stevenson B J, Farinelli L, Osteras M, Luescher I F. Highly diverse TCRα chain repertoire of pre-immune CD8⁺ T cells reveals new insights in gene recombination. EMBO J. 2012 Apr. 4; 31(7):1666-78; Robins H S, Campregher P V, Srivastava S K, Wacher A, Turtle C J, Kahsai O, Riddell S R, Warren E H, Carlson C S. Comprehensive assessment of T-cell receptor beta-chain diversity in alpha beta T cells. Blood. 2009 Nov. 5; 114(19):4099-107; Linnemann C, Heemskerk B, Kvistborg P, Kluin R J, Bolotin D A, Chen X, Bresser K, Nieuwland M, Schotte R, Michels S, Gomez-Eerland R, Jahn L, Hombrink P, Legrand N, Shu C J, Mamedov I Z, Velds A, Blank C U, Haanen J B, Turchaninova M A, Kerkhoven R M, Spits H, Hadrup S R, Heemskerk M H, Blankenstein T, Chudakov D M, Bendle G M, Schumacher T N. High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat Med. 2013 November; 19(11):1534-41; Turchaninova M A, Britanova O V, Bolotin D A, Shugay M, Putintseva E V, Staroverov D B, Sharonov G, Shcherbo D, Zvyagin I V, Mamedov I Z, Linnemann C, Schumacher T N, Chudakov D M. Pairing of T-cell receptor chains via emulsion PCR. Eur J Immunol. 2013 September; 43(9):2507-15.

Since most current TCR sequencing techniques require enrichment of TCR genes for sequencing, the majority of methods include an amplification step, in which the nucleic acids encoding the individual TCRs are amplified. Therefore, one of the challenges of the TCR sequencing relates to the ability of the technology to maintain the proportion of each TCR during the amplification. Thus, the ways in which TCR libraries are prepared have a strong impact on the quality and the reliability of the obtained sequencing results and on the conclusions than can be drawn from the data. Several approaches have been used to amplify and sequence TCR repertoires in the past, each method with its own set of issues.

One frequently employed method for TCR sequencing is based on a multiplex PCR step, in which all the primers for the V and the J segments are mixed together to amplify all the possible V(D)J rearrangements/combinations, as described in Robins H S, Campregher P V, Srivastava S K, Wacher A, Turtle C J, Kahsai O, Riddell S R, Warren E H, Carlson C S. Comprehensive assessment of T-cell receptor beta-chain diversity in alpha beta T cells. Blood. 2009 Nov. 5; 114(19):4099-107. The main drawback of this technology is that the amplification is not quantitative: Because the efficiency of each primer pair varies, some TCR sequences are preferentially represented in the library.

Another TCR sequencing method uses a process called “DNA gene capture” to isolate TCR encoding DNA fragments, as described in Linnemann C, Heemskerk B, Kvistborg P, Kluin R J, Bolotin D A, Chen X, Bresser K, Nieuwland M, Schotte R, Michels S, Gomez-Eerland R, Jahn L, Hombrink P, Legrand N, Shu C J, Mamedov I Z, Velds A, Blank C U, Haanen J B, Turchaninova M A, Kerkhoven R M, Spits H, Hadrup S R, Heemskerk M H, Blankenstein T, Chudakov D M, Bendle G M, Schumacher T N. High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat Med. 2013 November; 19(11):1534-41. However, since this method uses DNA rather than RNA, this method will also isolate V and J segments that have not yet undergone somatic rearrangement. As a consequence, many of the obtained sequencing data are uninformative for TCR gene identification as they do not contain the V(D)J region of rearranged TCR gene locus. Furthermore, using DNA instead of RNA for the TCR gene analysis may overestimate the diversity of the TCR repertoire as only one of the two β chains is expressed by the T-cells while the other gene is silenced (allelic exclusion).

A third method of TCR amplification is based on the 5′-Race PCR technology (SMARTer® Human TCR a/b Profiling Kit, Takara-Clontech). In this method, a nucleic acid adapter is added to the 5′-end of the cDNA during the reverse transcription step. As a result, TCR products can be subsequently amplified with a single primer pair, with one primer binding to the adapter at the 5′-end of the cDNA and the second primer binding to the constant region near the 3′-end of the cDNA. One of the disadvantages of this technique is that the amplification step will generate PCR fragments ranging between 500 and 600 bp. As the length of the V segment exceeds 400 bp it is actually not possible to sequence the V(D)J junction starting from the 5′end using Illumina® sequencing technology, which can generate sequencing reads of up to 300 bp only. Sequencing of the V/J junction is thus usually performed from the constant region, crossing the J segment, the CDR3 region and part of the V segment. However, sequencing errors increase with the length of the sequencing read, and are thus most frequently introduced in the V segments—the region most challenging to correctly assign due to the high homology between different V segments. Consequently, sequencing starting from the constant region may lead to a reduction in the number of V segments that can be identified unambiguously. While this caveat can be avoided by paired-end sequencing, such modification of the protocol will significantly increase the duration and cost associated with this method.

SUMMARY

With each of the current methods exhibiting significant shortcomings, there is thus a considerable need for a TCR sequencing technology that provides TCR repertoire data with high sensitivity and reliability.

Disclosed herein are methods and kits for sequencing of T-cell receptor repertoires and other immune cell repertoires, such as B-cell repertoires, with high sensitivity and reliability. In one embodiment, the methods include the steps of (1) providing RNA from T-cells, (2) transcribing RNA into complimentary RNA (cRNA), (3) reverse transcribing the cRNA into cDNA while introducing a common adapter to the 5′ end of the cDNA products, (4) amplifying the cDNA using a single primer pair, (5) further amplifying with PCR products with a single primer pair which introduces adapters for next generation sequencing, wherein the first primer binds to the common adapter region, and wherein the second primer binds to the constant region of the TCR gene, and (6) sequencing the PCR products. In one embodiment, the methods include the steps of (1) providing RNA from T-cells, (2) reverse transcribing the RNA into cDNA, (3) generating second strand cDNA while introducing a common adapter to the 5′ end of the cDNA products, (4) amplifying the cDNA using a single primer pair, (5) further amplifying with PCR products with a single primer pair which introduces adapters for next generation sequencing, wherein the first primer binds to the common adapter region, and wherein the second primer binds to the constant region of the TCR gene, and (6) sequencing the PCR products. These embodiments are also called SEQTR method (Sequencing T-cell Receptors). Also provided are kits containing primer mixtures for the sequencing of T-cell receptor repertoires. Similar methods and kits for sequencing of B-cell receptor repertoires are provided.

According to one aspect, methods for sequencing immune cell receptor genes are provided. The methods include (1) providing RNA from immune cells; 2)(a) optionally transcribing the RNA into complementary RNA (cRNA), followed by reverse transcribing the cRNA into complementary DNA (cDNA) using one or more primers that comprise a first adapter sequence, wherein each 5′ end of the cDNA produced by reverse transcription contains the first adapter sequence; (2)(b) if step (2)(a) is not performed, reverse transcribing the RNA into complementary DNA (cDNA), followed by transcribing the cDNA into second strand cDNA using one or more primers that comprise a first adapter sequence, wherein each 5′ end of the cDNA produced by transcribing the cDNA into second strand cDNA contains the first adapter sequence; (3) amplifying the cDNA to produce a first amplification product using a first primer pair comprising a first primer that hybridizes to the first adapter sequence and a second primer that hybridizes to a constant region of immune cell receptor gene; (4) amplifying the first amplification product to produce a second amplification product using a second primer pair, in which (i) a first primer of the second primer pair binds to the adapter sequence at the 5′ end of the second amplification product, (ii) the second primer of the second primer pair binds to the constant region of immune cell receptor gene in the second amplification product, and (iii) the first and second primers comprise adapter sequences for sequencing; and (5) sequencing the second amplification product.

In some embodiments, the reverse transcription step results in PCR products ranging from 150-600 bp. In some embodiments, the immune cell receptor genes are T-cell receptor (TCR) genes or B-cell receptor (BCR) genes.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR α chain V segments. In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) comprise one or more of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR β chain V segments. In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) comprise one or more of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR γ chain V segments.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR δ chain V segments.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to BCR heavy chain V segments.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to BCR light chain V segments.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) contain a nucleotide barcode sequence. In some embodiments, the nucleotide barcode comprises 6 to 20 nucleotides. In some embodiments, the nucleotide barcode consists of 9 nucleotides. In some embodiments, the nucleotide barcode consists of the sequence NNNNTNNNN, NNNNANNNN or HHHHHNNNN.

In some embodiments, the first adapter sequence of the one or more primers used for the reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) comprises a T7 adapter or an Illumina® adapter.

In some embodiments, the immune cells are T-cells and wherein the second primer of the first pair of primers hybridizes to the constant region of a TCR gene.

In some embodiments, the immune cells are B-cells and wherein the second primer of the first pair of primers hybridizes to the constant region of a BCR gene.

In some embodiments, the sequencing is next generation sequencing.

In some embodiments, the RNA from the immune cells is obtained by mixing immune cells with carrier cells before RNA extraction.

In some embodiments, the immune cells are tumor-infiltrating lymphocytes.

In some embodiments, the immune cells are CD4 or CD8 positive T-cells.

In some embodiments, the immune cells are purified from peripheral blood mononuclear cells (PBMC) before RNA extraction.

In some embodiments, the immune cells are part of a mixture of PBMC.

In some embodiments, the immune cells are derived from a mammal. In some embodiments, the mammal is a human or a mouse.

According to another aspect, kits for sequencing of T-cell receptors are provided. The kits include at least one primer which comprises a TCR α chain V segment portion of any one of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310 and a barcode sequence. In some embodiments, the kits include at least one primer including any one of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310.

According to another aspect, kits for sequencing of T-cell receptors are provided. The kits include at least one primer which comprises a TCR β chain V segment portion of any one of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360 and a barcode sequence. In some embodiments, the kits include at least one primer comprising any one of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360.

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the arrangement of (variable) V, diversity (D), joining (J) and constant (C) regions in the α and β chains of T-cell receptors. Figure taken from Murphy, K., Travers, P., Walport, M., & Janeway, C. (2012). Janeway's immunobiology. New York: Garland Science.

FIG. 2 is an illustration of three different TCR sequencing techniques that have been employed in the past.

FIG. 3 provides an overview of the SEQTR method, using TCR α chains as an example. Each bar represents a TCR α chain gene. In RNA and cRNA molecules, the order of the segments is, left to right: V segments, J segments, and the constant region. Barcode regions are added in cDNA molecule to the left of V segments; and T7 adapter regions are added to the left of the barcodes (also indicated by T7 primer amplification in PCR1 and PCR2 steps). Illumina® sequencing adapters are added in the PCR2 step to the 5′ and 3′ ends of the molecules, as shown in the last set of molecules.

FIG. 4 illustrates the sensitivity of the SEQTR method. 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively, were mixed with 5×10{circumflex over ( )}4 3T3 cells. The RNA was extracted and subjected to transcription, reverse transcription and one round of amplification (steps 2-4, see Detailed Description). The resulting PCR products were separated on an agarose gels and visualized with ethidium bromide.

FIG. 5 illustrates the specificity of the SEQTR method. 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively, were mixed with 5×10{circumflex over ( )}4 3T3 cells. The RNA was extracted and subjected to the SEQTR method. The percentages of sequencing reads that were or were not, respectively, associated with actual TCR genes are indicated.

FIG. 6 illustrates the unambiguous identification of TCR genes as a feature of the SEQTR method. 5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively. The RNA of each mixture was isolated and subjected to the SEQTR method. Reads that were not associated with TCR genes were removed from the data set. For the remaining reads, the percentages of reads that could or could not, respectively, be unambiguously assigned to specific V or J segments are indicated.

FIG. 7 illustrates the linearity of the SEQTR method. A fixed amount of DNA encoding a known TCR sequence was diluted at different concentrations into a DNA mixture representing a naïve CD8 T-cell repertoire. TCR repertoires of the individual mixtures were sequenced using the SEQTR method. The observed frequency of the known TCR sequence in the entire repertoire was plotted against the respective TCR gene dilution.

FIG. 8 illustrates the reproducibility of the SEQTR method. TCR repertoires were sequenced using the SEQTR method from one biological sample in two independent technical replicates. The frequencies for each V-J rearrangement/combination in TCR β chains were determined and compared between the two replicates. Each sphere represents a single V-J rearrangement with the size of a sphere indicating the relative frequency of the specific V-J recombination. Grey spheres represent rearrangements for which the relative frequencies detected in the two replicates differed by less than two-fold. Black spheres represent rearrangements for which the relative frequencies detected in the two replicates differed by more than two-fold.

FIGS. 9A-9C illustrates the diversity of three different TCR repertoire sequencing data set using the SEQTR method. FIG. 9A CD8 positive T-cells were isolated from peripheral blood mononuclear cells (PBMC), FIG. 9B additionally purified using tetramers conjugated with neo-epitope TEDYMIHII (SEQ ID NO:236) or FIG. 9C additionally purified using tetramer conjugated with neo-epitope (same as in FIG. 9B) and subsequently expanded in vitro. The TCR repertoires of the respective samples were sequenced using the SEQTR method and the relative frequencies of all observed V/J rearrangements/combinations plotted.

FIGS. 10A-10C illustrate the overlap of TCRs identified using a single cell cloning method and TCRs identified using the SEQTR method. FIG. 10A: T-cells were isolated from PBMC and subjected to an additional round of purification using tetramers conjugated with neo-epitope TEDYMIHII (SEQ ID NO: 236). The resulting cell population was then sorted by fluorescence-activated cell sorting (FACS). Half of the sorted cells were subjected to the SEQTR method to sequence the TCR repertoire. For the other half of cells, individual T-cell clones were isolated and expanded in vitro (single cell cloning). Once the clones were established, the TCR genes of each T-cell clones were amplified and sequenced using classical Sanger sequencing. FIG. 10B: The table shows all six TCRs identified using the single cell cloning method. The sequences correspond to SEQ ID NOs: 237 through 242 from top to bottom, respectively. FIG. 10C: The table shows the eight most frequent TCRs identified using the SEQTR method. The sequences correspond to SEQ ID NOs: 243 through 250 from top to bottom, respectively.

FIG. 11 illustrates the number of reads for different samples obtained using the TCR sequencing service “immunoSEQ®” offered by Adaptive Biotechnology. The requested number of reads per sample was 200,000 reads. The number on the x axis represent analysis of samples from 16 different patients. Columns, left to right, for each sample represent number of reads from: the tumor; the stroma (tissue surrounding the tumor); epitope specific TIL (Tumor Infiltrating Lymphocyte) stained with tetramer and sorted by FACS from the tumor sample (TET); and tetramer sorted TIL from a piece of the tumor that has been engrafted in a mice (mTET).

FIG. 12 illustrates the amplification of TCR genes from T-cells that are part of a PBMC mixture (upper panel) or from isolated, CD4 positive T-cells (lower panel), using steps 2 to 4 of the SEQTR method.

DETAILED DESCRIPTION

In light of the shortcomings of existing techniques to sequence TCRs, it was determined that a TCR sequencing technology providing the most reliable TCR repertoire data includes the following features:

-   -   1) The amplification of TCR genes is linear and does not employ         multiplex PCR, therefore avoiding artificial overrepresentation         of certain TCR sequences.     -   2) The method is based on RNA and not DNA, thus only providing         data for TCR sequences that have undergone rearrangement and         that are actually expressed in T-cells.     -   3) TCR genes are sequenced from the 5′ end, providing high         quality sequencing data and therefore maximizing reliable and         unambiguous identification of the highly homologous V segments.     -   4) Sequencing data include the highly variable CDR3 region,         therefore facilitating unambiguous identification of TCR         sequences.

The disclosed methods, systems and kits fulfill all these criteria. These same features are of use in sequencing receptors from other immune cells, such as B-cells.

In some embodiments, the immune cell receptor sequencing methods comprise the following steps:

-   -   (1) Providing total RNA (RNA) as the starting material;     -   (2)(a) Transcribing the RNA into complimentary RNA (cRNA)         followed by reverse transcribing the cRNA into cDNA, using         primers that introduce a common adapter to the 5′ end of the         cDNA products;     -   (2)(b) If step (2)(a) is not performed, reverse transcribing the         RNA into complementary DNA (cDNA), followed by transcribing the         cDNA into second strand cDNA using one or more primers that         comprise a first adapter sequence, wherein each 5′ end of the         cDNA produced by transcribing the cDNA into second strand cDNA         contains the first adapter sequence;     -   (3) Amplifying the cDNA products using a single primer pair;     -   (4) Amplifying the PCR products of step 4 using a single primer         pair, in which:         -   i. the primers introduce adapters for next generation             sequencing, and         -   ii. the first primer binds to the common adapter region at             the 5′ end of the PCR products, and         -   iii. the second primer binds to a region of the PCR products             that constitutes the constant region of the TCR to be             sequenced; and     -   (5) Sequencing the PCR products generated in step 4.

Genetic Information to be Sequenced

The genetic information to be sequenced is immune cell receptor genes. In the some embodiments of the invention, the genetic information to be sequenced comprises T-cell receptors genes. In some embodiments, the TCR genes that are sequenced encode TCR α chains or TCR β chains. In other embodiments, TCR genes that are sequenced encode TCR δ chains or TCR γ chains.

In other embodiments of the invention, the genetic information to be sequenced comprises B-cell receptor (BCR) genes.

Starting Material (Step 1)

RNA is isolated from immune cells and used to generate complimentary RNA (cRNA) by in vitro transcription. This is in contrast to existing TCR sequencing techniques that use DNA or complementary DNA (cDNA) as their genetic starting material.

In some embodiments, the immune cells from which RNA is obtained are isolated from peripheral blood mononuclear cells before RNA extraction. The immune cells are, in some embodiments, T-cells or B-cells.

In some embodiments, T-cells from which RNA is obtained express CD4 or CD8.

Generation of cRNA Through Transcription (Step (2)(a))

Complementary RNA (cRNA) is generated by in vitro transcription. Any method for performing in vitro transcription known to those skilled in molecular biology can be used. In some embodiments, the in vitro transcription in step 2 is performed using commercially available kits, such as the AMBION™ kits available from Thermo Fisher Scientific.

Reverse Transcription (Step (2)(a))

Reverse transcription of the cRNA is performed to generate complementary DNA (cDNA). Methods known to persons skilled in molecular biology are used to reverse transcribe cRNA to cDNA. Typically, such methods include hybridization of a primer to the 3′ end of the cRNA molecule and production of DNA starting at the hybridized primer using a reverse transcriptase enzyme and appropriate nucleotides, salts and buffers.

The choice of primers used in the reverse transcription reaction is important for the ability to differentiate between homologous, yet distinct, immune cell receptor sequences with high degrees of certainty and allows shortening of the V segments from the 5′ end, generating PCR products with a size of 250-300 bp. Such a size range of PCR products is optimal for next generation sequencing.

In some embodiments, the primers used for the reverse transcription are designed to bind within the V segments of the TCR genes (see FIG. 3). For example, the reverse transcription primers are designed to bind close enough to the V(D)J junction so that the resulting sequencing data cover the CDR3 of the V segment and the J segment, but far enough from the V(D)J junction to still allow differentiation between different V regions.

In some embodiments of the invention, a set of preferred primers is used (see, e.g., the sequences in Table 2 and Table 4, and Table 8 and Table 9). Due to the high degree of homology between different V segments, some of the primers described in Table 2 and Table 4 (and Table 8 and Table 9) bind to more than one V segment (see Table 3 and Table 5; the binding sites in their respective V segments for primers SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360 are indicated in Table 15 and Table 16). However, the design of the primers presented in Table 2 and Table 4 (likewise Table 8 and Table 9) still allows the unambiguous assignment/identification of the respective V segments based on differences between the V segments downstream of the primer-binding site. In an alternative embodiment of the invention, only a subset of the preferred primers SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360 may be used for the reverse transcription.

In yet another embodiment of the invention, primer sets may be used that bind to different regions in the V segments when compared to the primers having SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360. For instance, the binding site of one or more primers may be moved towards the CDR3 region of the TCR gene. Due to the high degree of homology between V segments, the further the primer binding site is moved in the direction of the CDR3 region of the TCR gene, the larger the likelihood that the resulting sequencing data are consistent with the presence of more than one V segment. While, in these cases, the respective V segments cannot be assigned or identified unambiguously, the number of V/J segments possibly present in the sample can often be narrowed down to a small subset. Depending on the application, such limited information can already be of value to the experimenter.

In another embodiment of the invention, the binding site of one or more primers may be moved towards the 5′ end of the V segment as compared to the binding sites of primers SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360. Many next generation sequencing technologies generate sequencing reads that are 150 bp long. Therefore, the further the primer binding site is moved towards the 5′ end of the V segment, the larger is the probability that the respective J segment (which can be found at the 3′ end of the resulting sequencing read) cannot be identified unambiguously. However, this problem can be circumvented by using alternative sequencing technologies that generate reads >150 bp.

In some embodiments, the primers used in step (2)(a) additionally contain a unique bar code. Such barcoding of each RNA molecule before the amplification can be used to correct the obtained sequencing results for PCR and sequencing errors.

In some embodiments, the primers for this reverse transcription step introduce a common T7 adapter at the 5′ end of the resulting PCR products. However, alternative adapter sequences are possible, including, but not limited to Illumina® adapters and sequences presented in Table 1.

TABLE 1 Examples for alternative nucleotide adapters that can be used instead of a T7 adapter sequence SEQ ID NO Primer name Primer sequence (5′ to 3′) 251 Original Eberwine AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGCT T7 252 Affymetrix T7 GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGT 253 Invitrogen T7 TAATACGACTCACTATAGGGAGGCGGT 254 Ambion T7 GGTAATACGACTCACTATAGGGAGAAGAGT 255 Agilent T7 AATTAATACGACTCACTATAGGGAGAT Reverse Transcription (Step (2)(b))

Reverse transcription of the RNA is performed to generate complementary DNA (cDNA). Methods known to persons skilled in molecular biology are used to reverse transcribe RNA to cDNA. Typically, such methods include hybridization of a primer to the 3′ end of the RNA molecule and production of DNA starting at the hybridized primer using a reverse transcriptase enzyme and appropriate nucleotides, salts and buffers.

Transcribing the cDNA into Second Strand cDNA (Step (2)(b))

Following generation of cDNA, second strand cDNA is synthesized using methods known to persons skilled in molecular biology. Typically, such methods include hybridization of a primer to the 3′ end of the cDNA molecule and production of second strand cDNA starting at the hybridized primer using a polymerase enzyme and appropriate nucleotides, salts and buffers.

The choice of primers used in the second strand synthesis reaction is step (2)(b) is as described above for reverse transcription in step (2)(a). The choice of primers is important for the ability to differentiate between homologous, yet distinct, immune cell receptor sequences with high degrees of certainty and allows shortening of the V segments from the 5′ end, generating PCR products with a size of 250-300 bp. Such a size range of PCR products is optimal for next generation sequencing.

Amplification (Step 3)

Amplification of the cDNA is performed by any of the well-known amplification reactions, such as polymerase chain reaction (PCR). Methods known to persons skilled in the molecular biology art are used to amplify the cDNA or a portion thereof (e.g., as depicted in FIG. 3). Typically, such methods include hybridization of a pair of primers to the cDNA molecule and amplification of the DNA sequence between the hybridized primers using a polymerase enzyme and appropriate nucleotides, salts and buffers.

In some embodiments, the first primer of a primer pair used in an amplification step binds to the common adapter region of the cDNA products produced in step 3 and the second primer of the primer pair binds to a region of the cDNA products that constitutes the constant region of the TCR to be sequenced (see FIG. 3).

Of note, not all reverse primers designed to target the constant region of the TCR gene perform equally well in this reaction. For example, the primers listed in Table 7 all failed to provide good amplification with the selected T7 5′ adapter. Therefore, in certain embodiments, the primers listed in are Table 6 used in this amplification step.

Amplification (Step 4)

A second amplification step is performed to add additional sequences to the amplified molecules, such as sequences that are useful in downstream DNA sequencing reactions. In some embodiments of the present invention, the primers used in this step add appropriate adapters for Illumina® sequencing.

Sequencing (Step 5)

Various suitable sequencing methods described herein or known in the art are used to obtain sequence information from the amplified sequences from the nucleic acid molecules within a sample. For example, sequencing methodologies that can be used in the methods disclosed herein include: classic Sanger sequencing, massively parallel sequencing, next generation sequencing, polony sequencing, 454 pyrosequencing, Illumina® sequencing, Solexa® sequencing, SOLiD™ sequencing, ion semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real time sequencing, nanopore DNA sequencing, tunneling currents DNA sequencing, sequencing by hybridization, sequencing with mass spectrometry, microfluidic Sanger sequencing, microscopy-based sequencing, RNA polymerase sequencing, in vitro virus high-throughput sequencing, Maxam-Gilbert sequencing, single-end sequencing, paired-end sequencing, deep sequencing, and/or ultra-deep sequencing.

Definitions

As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

As used herein, a “primer” is a nucleic acid molecule that hybridizes with a complementary (including partially complementary) polynucleotide strand. Primers can be DNA molecules, RNA molecules, or DNA or RNA analogs. DNA or RNA analogs can be synthesized from nucleotide analogs.

Examples Example 1: Exemplary Protocol for the SEQTR Method Using T7 and TrueSeq Adapters

TCR α and β chain genes were sequenced in two independent reactions.

1) Starting Material and RNA Extraction

-   -   To obtain sufficient amounts of RNA in the extraction, a minimum         of 500,000 T-cells were used as starting material.         Alternatively, and especially in instances where fewer T-cells         were available, T-cells were mixed with 50,000 mouse 3T3 cells         that served as carrier. T-cell RNA was extracted using the         RNeasy® Micro Kit from Qiagen Inc. according the manufacturer's         instruction with the following modification: Elution was         performed with 20 μl of water preheated to 50° C. RNA quality         and quantity was verified using a fragment analyzer.

2) cRNA Synthesis by In Vitro Transcription (IVT):

-   -   In vitro transcription of isolated RNA was performed using the         MessageAmp™ II aRNA Amplification Kit from Ambion® (Thermo         Fisher Scientific), which contains enzymes, buffers and         nucleotides required to perform the first and second strand cDNA         and the in vitro transcription. The kit also provides all         columns and reagents needed for the cDNA and cRNA purifications.         RNA amplification was performed according to the manufacturer's         instructions with the following modifications: 1) Between 0.5         and 1 μg of total RNA as was used as starting material. 2) The         IVT was performed in a final volume of 40 μl, and incubated at         37° C. for 16 h. Purified cRNA was quantified by absorbance         using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific).

3) cDNA Synthesis by Reverse Transcription:

-   -   The reverse transcription of the cRNA was performed with the         SuperScript® III from Invitrogen (Thermo Fisher Scientific). The         kit provides the enzyme, the buffer and the dithiothreitol (DTT)         needed for the reaction. Deoxynucleotides (dNTPs) and RNAsin®         Ribonuclease inhibitor were purchased from Promega. The         sequences for the primers used for the reverse transcription can         be found in Table 2 (primers for sequencing TCR α chain genes)         and Table 4 (primers for sequencing TCR (3 chain genes).     -   500 ng of cRNA were used as starting material for the reverse         transcription. cRNA was mixed with 1 μl hTRAV or hTRBV primers         mix (2 μM each) and 1 μl dNTP (25 mM) in a final volume of 13         μl. The mix was first incubated at 70° C. for 10 min, then at         50° C. for 30 s. 4 μl 5× buffer, 1 μl DTT (100 mM), 1 μl         SuperScript III and 1 μl RNAsin® were added to the mix. The         samples were subsequently incubated for at 55° C. 1 h and then         at 85° C. for 5 min. After the cDNA synthesis, 1 μg DNase-free         RNase (Roche) was added to the cDNA and incubated at 37° C. for         30 min to remove the cRNA.

4) TCR Gene Amplification:

-   -   TCR gene amplification was performed using a Phusion®         High-Fidelity DNA polymerase (New England Biolabs) under the         following conditions:     -   PCR mix: 1 μl cDNA from step 3, 1 μl dNTPs (25 mM), 1 μl primer         mix (10 μM each, see Table 5), 5 μl 5× buffer and 0.2 μl         Phusion® enzyme in a total volume of 25 μl.     -   PCR conditions:         -   94° C. for 5 min         -   20 to 30 cycles of             -   98° C. for 10 s             -   55° C. for 30 s             -   72° C. for 30 s         -   72° C. for 2 min     -   PCR products were purified either from agarose gels (using a         Qiaquick Gel Extraction Kit from Qiagen) or using an ExoSAP-IT®         PCR Product Cleanup Kit (Affymetrix) according to the         manufacturer's instructions.

5) Addition of Next Generation Sequencing Adapters:

-   -   Illumina® sequencing adapters were added by PCR using a Phusion®         High-Fidelity DNA polymerase (New England Biolabs). One third of         the purified PCR product obtained in step 4 was mixed with 0.5         μl dNTPs (25 mM), 1 μl primer mix (10 μM each, see Table 8), 5         μl 5× buffer and 0.2 μl Phusion® enzyme in a total volume of 25     -   PCR conditions:         -   94° C. for 5 min         -   perform 12 cycles of:             -   98° C. for 10 s             -   55° C. for 30 s             -   72° C. for 30 s         -   72° C. for 2 min

6) TCR Library Purification:

-   -   10 μl of the PCR product from step 5 were purified using an         ExoSAP-IT® PCR Product Cleanup Kit (Affymetrix) or Ampure XP         beads (Beckman Coulter) according to the manufacturer's         instruction. Samples could then directly be used for Illumina®         sequencing.

TABLE 2 Preferred primer sequences for amplification of TCR α chain V segments. N can be any nucleotide. The sequences for primers presented in this table consist of three parts (listed from 5′ to 3′): T7 adapter, barcode and TCR α chain V segment. SEQ Sequence Sequence ID Primer T7 adapter portion  barcode portion Sequence TCR α chain V segment NO name of the primer of the primer portion of the primer  1 hTRAV1-1 TGTAATACGACTCACTATAG NNNNTNNNN CTTCTACAGGAGCTCCAGATGAAAG  2 hTRAV1-2 TGTAATACGACTCACTATAG NNNNTNNNN CTTTTGAAGGAGCTCCAGATGAAAG  3 hTRAV2 TGTAATACGACTCACTATAG NNNNTNNNN TGCTCATCCTCCAGGTGCGGGA  4 hTRAV3 TGTAATACGACTCACTATAG NNNNTNNNN GAAGAAACCATCTGCCCTTGTGA  5 hTRAV4 TGTAATACGACTCACTATAG NNNNTNNNN CCTGCCCCGGGTTTCCCTGAGCGAC  6 hTRAV5 TGTAATACGACTCACTATAG NNNNTNNNN TCTCTGCGCATTGCAGACACCCA  7 hTRAV6 TGTAATACGACTCACTATAG NNNNTNNNN TTGTTTCATATCACAGCCTCCCA  8 hTRAV7 TGTAATACGACTCACTATAG NNNNTNNNN GCTTGTACATTACAGCCGTGCA  9 hTRAV8-1/8-3 TGTAATACGACTCACTATAG NNNNTNNNN ATCTGAGGAAACCCTCTGTGCA 10 hTRAV8-2/8-4 TGTAATACGACTCACTATAG NNNNTNNNN ACCTGACGAAACCCTCAGCCCAT 11 hTRAV8-5 TGTAATACGACTCACTATAG NNNNTNNNN CCTATGCCTGTCTTTACTTTAATC 12 hTRAV8-6 TGTAATACGACTCACTATAG NNNNTNNNN CTTGAGGAAACCCTCAGTCCATAT 13 hTRAV8-7 TGTAATACGACTCACTATAG NNNNTNNNN GAAACCATCAACCCATGTGAGTGA 14 hTRAV9-1 TGTAATACGACTCACTATAG NNNNTNNNN ACTTGGAGAAAGACTCAGTTCAA 15 hTRAV9-2 TGTAATACGACTCACTATAG NNNNTNNNN ACTTGGAGAAAGGCTCAGTTCAA 16 hTRAV10 TGTAATACGACTCACTATAG NNNNTNNNN CTGCACATCACAGCCTCCCA 17 hTRAV11 TGTAATACGACTCACTATAG NNNNTNNNN GTTTGGAATATCGCAGCCTCTCAT 18 hTRAV12-1 TGTAATACGACTCACTATAG NNNNTNNNN CCCTGCTCATCAGAGACTCCAAG 19 hTRAV12-2 TGTAATACGACTCACTATAG NNNNTNNNN CTCTGCTCATCAGAGACTCCCAG 20 hTRAV12-3 TGTAATACGACTCACTATAG NNNNTNNNN CCTTGTTCATCAGAGACTCACAG 21 hTRAV13-1 TGTAATACGACTCACTATAG NNNNTNNNN TCCCTGCACATCACAGAGACCCAA 22 hTRAV13-2 TGTAATACGACTCACTATAG NNNNTNNNN TCTCTGCAAATTGCAGCTACTCAA 23 hTRAV14 TGTAATACGACTCACTATAG NNNNTNNNN TTGTCATCTCCGCTTCACAACTGG 24 hTRAV15 TGTAATACGACTCACTATAG NNNNTNNNN GTTTTGAATATGCTGGTCTCTCAT 25 hTRAV16 TGTAATACGACTCACTATAG NNNNTNNNN CCTGAAGAAACCATTTGCTCAAGA 26 hTRAV17 TGTAATACGACTCACTATAG NNNNTNNNN TCCTTGTTGATCACGGCTTCCCGG 27 hTRAV18 TGTAATACGACTCACTATAG NNNNTNNNN ACCTGGAGAAGCCCTCGGTGCA 28 hTRAV19 TGTAATACGACTCACTATAG NNNNTNNNN CACCATCACAGCCTCACAAGTCGT 29 hTRAV20 TGTAATACGACTCACTATAG NNNNTNNNN TTTCTGCACATCACAGCCCCTA 30 hTRAV21 TGTAATACGACTCACTATAG NNNNTNNNN CTTTATACATTGCAGCTTCTCAGCC 31 hTRAV22 TGTAATACGACTCACTATAG NNNNTNNNN GTACATTTCCTCTTCCCAGACCAC 32 hTRAV23 TGTAATACGACTCACTATAG NNNNTNNNN CATTGCATATCATGGATTCCCAGC 33 hTRAV24 TGTAATACGACTCACTATAG NNNNTNNNN GCTATTTGTACATCAAAGGATCCC 34 hTRAV25 TGTAATACGACTCACTATAG NNNNTNNNN CAGCTCCCTGCACATCACAGCCA 35 hTRAV26-1 TGTAATACGACTCACTATAG NNNNTNNNN TTGATCCTGCCCCACGCTACGCTGA 36 hTRAV26-2 TGTAATACGACTCACTATAG NNNNTNNNN TTGATCCTGCACCGTGCTACCTTGA 37 hTRAV27 TGTAATACGACTCACTATAG NNNNTNNNN GTTCTCTCCACATCACTGCAGCC 38 hTRAV28 TGTAATACGACTCACTATAG NNNNTNNNN GCCACCTATACATCAGATTCCCA 39 hTRAV29 TGTAATACGACTCACTATAG NNNNTNNNN TCTCTGCACATTGTGCCCTCCCA 40 hTRAV30 TGTAATACGACTCACTATAG NNNNTNNNN CCCTGTACCTTACGGCCTCCCAGCT 41 hTRAV31 TGTAATACGACTCACTATAG NNNNTNNNN CTTATCATATCATCATCACAGCCA 42 hTRAV32 TGTAATACGACTCACTATAG NNNNTNNNN TCCCTGCATATTACAGCCACCCAA 43 hTRAV33 TGTAATACGACTCACTATAG NNNNTNNNN ACCTCACCATCAATTCCTTAAAAC 44 hTRAV34 TGTAATACGACTCACTATAG NNNNTNNNN TCCCTGCATATCACAGCCTCCCAG 45 hTRAV35 TGTAATACGACTCACTATAG NNNNTNNNN CTTCCTGAATATCTCAGCATCCAT 46 hTRAV36 TGTAATACGACTCACTATAG NNNNTNNNN TCCTGAACATCACAGCCACCCAG 47 hTRAV37 TGTAATACGACTCACTATAG NNNNTNNNN TCCCTGCACATACAGGATTCCCAG 48 hTRAV38 TGTAATACGACTCACTATAG NNNNTNNNN CAAGATCTCAGACTCACAGCTGG 49 hTRAV39 TGTAATACGACTCACTATAG NNNNTNNNN CCGTCTCAGCACCCTCCACATCA 50 hTRAV40 TGTAATACGACTCACTATAG NNNNTNNNN CCATTGTGAAATATTCAGTCCAGG

TABLE 3 V segments targeted by each primer used for the amplification of TCR α chain V segments. SEQ ID NO Primer Targeted V segment(s) 1 hTRAV1-1 hTRAV01-1 2 hTRAV1-2 hTRAV01-2 3 hTRAV2 hTRAV02 4 hTRAV3 hTRAV03 5 hTRAV4 hTRAV04 6 hTRAV5 hTRAV05 7 hTRAV6 hTRAV06 8 hTRAV7 hTRAV07 9 hTRAV8-1/8-3 hTRAV08-1, hTRAV08-3 10 hTRAV8-2/8-4 hTRAV08-2, hTRAV08-4 11 hTRAV8-5 hTRAV08-5 12 hTRAV8-6 hTRAV08-6 13 hTRAV8-7 hTRAV08-7 14 hTRAV9-1 hTRAV09-1 15 hTRAV9-2 hTRAV09-2 16 hTRAV10 hTRAV10, hTRAV41 17 hTRAV11 hTRAV11 18 hTRAV12-1 hTRAV12-1 19 hTRAV12-2 hTRAV12-2 20 hTRAV12-3 hTRAV12-3 21 hTRAV13-1 hTRAV13-1 22 hTRAV13-2 hTRAV13-2 23 hTRAV14 hTRAV14 24 hTRAV15 hTRAV15 25 hTRAV16 hTRAV16 26 hTRAV17 hTRAV17 27 hTRAV18 hTRAV18 28 hTRAV19 hTRAV19 29 hTRAV20 hTRAV20 30 hTRAV21 hTRAV21 31 hTRAV22 hTRAV22 32 hTRAV23 hTRAV23 33 hTRAV24 hTRAV24 34 hTRAV25 hTRAV25 35 hTRAV26-1 hTRAV26-1 36 hTRAV26-2 hTRAV26-2 37 hTRAV27 hTRAV27 38 hTRAV28 hTRAV28 39 hTRAV29 hTRAV29 40 hTRAV30 hTRAV30 41 hTRAV31 hTRAV31 42 hTRAV32 hTRAV32 43 hTRAV33 hTRAV33 44 hTRAV34 hTRAV34 45 hTRAV35 hTRAV35 46 hTRAV36 hTRAV36 47 hTRAV37 hTRAV37 48 hTRAV38 hTRAV38-1, hTRAV38-2 49 hTRAV39 hTRAV39 50 hTRAV40 hTRAV40

TABLE 4 Preferred primer sequences for amplification of TCR β chain V segments. N can be any nucleotide. The sequences for primers presented in this table consist of three parts (listed from 5′ to 3′): T7 adapter, barcode and TCR β chain V segment. SEQ Sequence Sequence ID Primer T7 adapter portion  barcode portion Sequence TCR β chain V segment NO name of the primer of the primer portion of the primer  51 hTRBV1 TGTAATACGACTCACTATAG NNNNANNNN GTGGTCGCACTGCAGCAAGAAGA  52 hTRBV2 TGTAATACGACTCACTATAG NNNNANNNN GATCCGGTCCACAAAGCTGGAGGA  53 hTRBV3-1 TGTAATACGACTCACTATAG NNNNANNNN CATCAATTCCCTGGAGCTTGGTGA  54 hTRBV4-1 TGTAATACGACTCACTATAG NNNNANNNN TTCACCTACACGCCCTGCAGCCAG  55 hTRBV4-2 TGTAATACGACTCACTATAG NNNNANNNN TTCACCTACACACCCTGCAGCCAG  56 hTRBV5-1 TGTAATACGACTCACTATAG NNNNANNNN GAATGTGAGCACCTTGGAGCTGG  57 hTRBV5-2 TGTAATACGACTCACTATAG NNNNANNNN TACTGAGTCAAACACGGAGCTAGG  58 hTRBV5-3 TGTAATACGACTCACTATAG NNNNANNNN GCTCTGAGATGAATGTGAGTGCCT  59 hTRBV5-4 TGTAATACGACTCACTATAG NNNNANNNN CTGAGCTGAATGTGAACGCCTT  60 hTRBV6-1 TGTAATACGACTCACTATAG NNNNANNNN GAGTTCTCGCTCAGGCTGGAGT  61 hTRBV6-2 TGTAATACGACTCACTATAG NNNNANNNN CTGGGGTTGGAGTCGGCTGCTC  62 hTRBV6-4 TGTAATACGACTCACTATAG NNNNANNNN CCCCTCACGTTGGCGTCTGCTG  63 hTRBV6-5 TGTAATACGACTCACTATAG NNNNANNNN TCCCGCTCAGGCTGCTGTCGGC  64 hTRBV6-6 TGTAATACGACTCACTATAG NNNNANNNN GATTTCCCGCTCAGGCTGGAGT  65 hTRBV6-7 TGTAATACGACTCACTATAG NNNNANNNN TCCCCCTCAAGCTGGAGTCAGCT  66 hTRBV6-8 TGTAATACGACTCACTATAG NNNNANNNN TCCCACTCAGGCTGGTGTCGGC  67 hTRBV7-1 TGTAATACGACTCACTATAG NNNNANNNN CTCTGAAGTTCCAGCGCACACA  68 hTRBV7-2 TGTAATACGACTCACTATAG NNNNANNNN GATCCAGCGCACACAGCAGGAG  69 hTRBV7-3 TGTAATACGACTCACTATAG NNNNANNNN ACTCTGAAGATCCAGCGCACAGA  70 hTRBV7-5 TGTAATACGACTCACTATAG NNNNANNNN AGATCCAGCGCACAGAGCAAGG  71 hTRBV7-6 TGTAATACGACTCACTATAG NNNNANNNN CAGCGCACAGAGCAGCGGGACT  72 hTRBV7-9 TGTAATACGACTCACTATAG NNNNANNNN GAGATCCAGCGCACAGAGCAGG  73 hTRBV8-1 TGTAATACGACTCACTATAG NNNNANNNN CCCTCAACCCTGGAGTCTACTA  74 hTRBV8-2 TGTAATACGACTCACTATAG NNNNANNNN TCCCCAATCCTGGCATCCACCA  75 hTRBV9 TGTAATACGACTCACTATAG NNNNANNNN CTAAACCTGAGCTCTCTGGAGCT  76 hTRBV10-1 TGTAATACGACTCACTATAG NNNNANNNN CCCTCACTCTGGAGTCTGCTGC  77 hTRBV10-2 TGTAATACGACTCACTATAG NNNNANNNN CCCTCACTCTGGAGTCAGCTAC  78 hTRBV10-3 TGTAATACGACTCACTATAG NNNNANNNN TCCTCACTCTGGAGTCCGCTAC  79 hTRBV11-1 TGTAATACGACTCACTATAG NNNNANNNN CCACTCTCAAGATCCAGCCTGCA  80 hTRBV12-1 TGTAATACGACTCACTATAG NNNNANNNN GAGGATCCAGCCCATGGAACCCA  81 hTRBV12-2 TGTAATACGACTCACTATAG NNNNANNNN CTGAAGATCCAGCCTGCAGAGC  82 hTRBV12-3 TGTAATACGACTCACTATAG NNNNANNNN CAGCCCTCAGAACCCAGGGACT  83 hTRBV13 TGTAATACGACTCACTATAG NNNNANNNN GAGCTCCTTGGAGCTGGGGGACT  84 hTRBV14 TGTAATACGACTCACTATAG NNNNANNNN GGTGCAGCCTGCAGAACTGGAG  85 hTRBV15 TGTAATACGACTCACTATAG NNNNANNNN GACATCCGCTCACCAGGCCTGG  86 hTRBV16 TGTAATACGACTCACTATAG NNNNANNNN TGAGATCCAGGCTACGAAGCTT  87 hTRBV17 TGTAATACGACTCACTATAG NNNNANNNN GAAGATCCATCCCGCAGAGCCG  88 hTRBV18 TGTAATACGACTCACTATAG NNNNANNNN GGATCCAGCAGGTAGTGCGAGG  89 hTRBV19 TGTAATACGACTCACTATAG NNNNANNNN CACTGTGACATCGGCCCAAAAG  90 hTRBV20 TGTAATACGACTCACTATAG NNNNANNNN CTGACAGTGACCAGTGCCCATC  91 hTRBV21 TGTAATACGACTCACTATAG NNNNANNNN GAGATCCAGTCCACGGAGTCAG  92 hTRBV22 TGTAATACGACTCACTATAG NNNNANNNN GTGAAGTTGGCCCACACCAGCCA  93 hTRBV23 TGTAATACGACTCACTATAG NNNNANNNN CCTGGCAATCCTGTCCTCAGAA  94 hTRBV24 TGTAATACGACTCACTATAG NNNNANNNN GAGTCTGCCATCCCCAACCAGA  95 hTRBV25 TGTAATACGACTCACTATAG NNNNANNNN GGAGTCTGCCAGGCCCTCACA  96 hTRBV26 TGTAATACGACTCACTATAG NNNNANNNN GAAGTCTGCCAGCACCAACCAG  97 hTRBV27 TGTAATACGACTCACTATAG NNNNANNNN GGAGTCGCCCAGCCCCAACCAG  98 hTRBV28 TGTAATACGACTCACTATAG NNNNANNNN GGAGTCCGCCAGCACCAACCAG  99 hTRBV29 TGTAATACGACTCACTATAG NNNNANNNN GTGAGCAACATGAGCCCTGAAGA 100 hTRBV30 TGTAATACGACTCACTATAG NNNNANNNN GAGTTCTAAGAAGCTCCTTCTCA

TABLE 5 V segments targeted by each primer used for the amplification of TCR β chain V segments. TCR b chain V SEQ ID NO segment name Targeted V segment(s) 51 hTRBV1 hTRBV01 52 hTRBV2 hTRBV02 53 hTRBV3-1 hTRBV03-1, hTRBV03-2 54 hTRBV4-1 hTRBV04-1 55 hTRBV4-2 hTRBV04-2, hTRBV04-3 56 hTRBV5-1 hTRBV05-1 57 hTRBV5-2 hTRBV05-2 58 hTRBV5-3 hTRBV05-3 59 hTRBV5-4 hTRBV05-4, hTRBV05-5, hTRBV05-6, hTRBV05-7, hTRBV05-8 60 hTRBV6-1 hTRBV06-1 61 hTRBV6-2 hTRBV06-2, hTRBV06-3 62 hTRBV6-4 hTRBV06-4 63 hTRBV6-5 hTRBV06-5 64 hTRBV6-6 hTRBV06-6, hTRBV06-9 65 hTRBV6-7 hTRBV06-7 66 hTRBV6-8 hTRBV06-8 67 hTRBV7-1 hTRBV07-1 68 hTRBV7-2 hTRBV07-2, hTRBV07-8 69 hTRBV7-3 hTRBV07-3, hTRBV07-4 70 hTRBV7-5 hTRBV07-5 71 hTRBV7-6 hTRBV07-6, hTRBV07-7 72 hTRBV7-9 hTRBV07-9 73 hTRBV8-1 hTRBV08-1 74 hTRBV8-2 hTRBV08-2 75 hTRBV9 hTRBV09 76 hTRBV10-1 hTRBV10-1 77 hTRBV10-2 hTRBV10-2 78 hTRBV10-3 hTRBV10-3 79 hTRBV11-1 hTRBV11-1, hTRBV11-2, hTRBV11-3 80 hTRBV12-1 hTRBV12-1 81 hTRBV12-2 hTRBV12-2 82 hTRBV12-3 hTRBV12-3, hTRBV12-4, hTRBV12-5 83 hTRBV13 hTRBV13 84 hTRBV14 hTRBV14 85 hTRBV15 hTRBV15 86 hTRBV16 hTRBV16 87 hTRBV17 hTRBV17 88 hTRBV18 hTRBV18 89 hTRBV19 hTRBV19 90 hTRBV20 hTRBV20 91 hTRBV21 hTRBV21 92 hTRBV22 hTRBV22 93 hTRBV23 hTRBV23 94 hTRBV24 hTRBV24 95 hTRBV25 hTRBV25 96 hTRBV26 hTRBV26 97 hTRBV27 hTRBV27 98 hTRBV28 hTRBV28 99 hTRBV29 hTRBV29 100 hTRBV30 hTRBV30

TABLE 6 Primers for TCR gene amplification. Primer pair for sequencing of TCR α genes: SEQ ID NO 101 and 102. Primer pair for sequencing of TCR  β genes: SEQ ID NO 101 and 103. SEQ ID NO Primer name Primer sequence TCR chain 101 Forward primer T7 TRAV/TRBV TGTAATACGACTCACTATAG α and β 102 Reverse primer PCR 1 TRAV GGCCACAGCACTGTTGCTCTTGAAG α 103 Reverse primer PCR 1 TRABV CCACTGTGCACCTCCTTCCCATTC β

TABLE 7 Reverse primers for TCR gene amplification that did not result in successful amplification of PCR products. SEQ ID NO Primer sequence TCR chain 104 TCGACCAGCTTGACATCACAGG α 105 CAGATTTGTTGCTCCAGGCCACAG α 106 TCTGTGATATACACATCAGAATC α 107 GAATCAAAATCGGTGAATAGGCAG α 108 GGCAGACAGACTTGTCACTGGATT α 109 TAGGACACCGAGGTAAAGCCAC β 110 CTGGGTGACGGGTTTGGCCCTAT β 111 TTGACAGCGGAAGTGGTTGC β 112 GGCTGCTCAGGCAGTATCTGGAGTC β 113 GCCAGGCACACCAGTGTGGCCTTTT β

TABLE 8 Primers for addition of Next Generation Sequencing adapters. The primer portion corresponding to the Illumina ® adapters (forward and reverse) is underlined in forward and reverse primers shown below. Primer pair for sequencing of TCR α genes: SEQ ID NOS: 114 and 115. Primer pair for sequencing of TCR β genes:  SEQ ID NOS: 114 and 116. SEQ ID TCR NO Primer name Primer sequence chain 114 Forward primer AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCT α and β Illumina_T7 CTTCCGATCTTGTAATACGACTCACTATAG TRAV/TRBV 115 Reverse primer CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGAC α PCR 2 TRAC GTGTGCTCTTCCGATCCTCAGCTGGTACACGGCAGGGTCA 116 Reverse primer CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGAC β PCR 2 TRBC GTGTGCTCTTCCGATCAAACACAGCGACCTCGGGTGGGAAC

Example 2: Exemplary Protocol for the SEQTR Method Using Nextera Adapters

TCR α and β chain genes were sequenced in two independent reactions.

1) Starting Material and RNA Extraction

-   -   To obtain sufficient amounts of RNA in the extraction, a minimum         of 500,000 T-cells were used as starting material.         Alternatively, and especially in instances where fewer T-cells         were available, T-cells were mixed with 50,000 mouse 3T3 cells         that served as carrier. T-cell RNA was extracted using the         RNeasy® Micro Kit from Qiagen Inc. according the manufacturer's         instruction with the following modification: Elution was         performed with 20 μl of water preheated to 50° C. RNA quality         and quantity was verified using a fragment analyzer.

2) cRNA Synthesis by In Vitro Transcription (IVT):

-   -   In vitro transcription of isolated RNA was performed using the         MessageAmp™ II aRNA Amplification Kit from Ambion® (Thermo         Fisher Scientific), which contains enzymes, buffers and         nucleotides required to perform the first and second strand cDNA         and the in vitro transcription. The kit also provides all         columns and reagents needed for the cDNA and cRNA purifications.         RNA amplification was performed according to the manufacturer's         instructions with the following modifications:     -   1) Between 0.5 and 1 μg of total RNA as was used as starting         material. 2) The IVT was performed in a final volume of 40 μl,         and incubated at 37° C. for 16 h. Purified cRNA was quantified         by absorbance using a NanoDrop™ spectrophotometer (Thermo Fisher         Scientific).

3) cDNA Synthesis by Reverse Transcription:

-   -   The reverse transcription of the cRNA was performed with the         SuperScript® III from Invitrogen (Thermo Fisher Scientific). The         kit provides the enzyme, the buffer and the dithiothreitol (DTT)         needed for the reaction. Deoxynucleotides (dNTPs) and RNAsin®         Ribonuclease inhibitor were purchased from Promega. The         sequences for the primers used for the reverse transcription can         be found in Table 9 (primers for sequencing TCR α chain genes)         and Table 10 (primers for sequencing TCR β chain genes).     -   500 ng of cRNA were used as starting material for the reverse         transcription. cRNA was mixed with 1 μl hTRAV or hTRBV primers         mix (2 μM each) and 1 μl dNTP (25 mM) in a final volume of 13         μl. The mix was first incubated at 70° C. for 10 min, then at         50° C. for 30 s. 4 μl 5× buffer, 1 μl DTT (100 mM), 1 μl         SuperScript III and 1 μl RNAsin® were added to the mix. The         samples were subsequently incubated for at 55° C. 1 h and then         at 85° C. for 5 min. After the cDNA synthesis, 1 μg DNase-free         RNase (Roche) was added to the cDNA and incubated at 37° C. for         30 min to remove the cRNA.

4) TCR Gene Amplification:

-   -   TCR gene amplification was performed using a Phusion®         High-Fidelity DNA polymerase (New England Biolabs) under the         following conditions:     -   PCR mix: 1 μl cDNA from step 3, 0.4 μl dNTPs (10 mM), 0.4 μl         primer mix (20 μM Nextera5′, 10 μM Reverse primer PCR 1 TRAV or         2.5 μM Reverse primer PCR1 TRBV, see Table 11), 2 μl 5× buffer         and 0.2 μl Phusion® enzyme in a total volume of 10 μl.     -   PCR conditions:         -   94° C. for 5 min         -   20 cycles of             -   98° C. for 10 s             -   55° C. for 30 s             -   72° C. for 30 s         -   72° C. for 2 min     -   PCR products were purified using 1 μl of ExoSAP-IT® PCR Product         Cleanup Kit (Affymetrix) according to the manufacturer's         instructions.

5) Addition of Next Generation Sequencing Adapters:

-   -   Illumina® sequencing adapters were added by PCR using a Phusion®         High-Fidelity DNA polymerase (New England Biolabs). The         following mix was added to the 11 μl of PCR1: 1 μl dNTPs (10         mM), 1 μl primer mix (1.25 μM each, see Table 12), 3 μl 5×         buffer and 0.2 μl Phusion® enzyme and 9.8 μl of H₂O.     -   PCR conditions:         -   94° C. for 5 min         -   perform 25 cycles of:             -   98° C. for 10 s             -   55° C. for 30 s             -   72° C. for 30 s         -   72° C. for 2 min

6) TCR Library Purification:

-   -   10 μl of the PCR product from step 5 were purified using an         AMPURE XP beads (Beckman Coulter) according to the         manufacturer's instruction. Samples could then directly be used         for Illumina® sequencing.

TABLE 9 Preferred primer sequences for amplification of TCR α chain V segments. N can be any nucleotide. The sequences for primers presented in this table consist of three parts (listed from 5′ to 3′): T7 adapter, barcode and TCR α chain V segment. SEQ Sequence Nextera Sequence ID Primer adapter portion  barcode portion Sequence TCR α chain V segment NO name of the primer of the primer portion of the primer 261 hTRAV1-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTCTACAGGAGCTCCAGATGAAAG GTATAAGAGACAG 262 hTRAV1-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTTTGAAGGAGCTCCAGATGAAAG GTATAAGAGACAG 263 hTRAV2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TGCTCATCCTCCAGGTGCGGGA GTATAAGAGACAG 264 hTRAV3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAAGAAACCATCTGCCCTTGTGA GTATAAGAGACAG 265 hTRAV4 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTGCCCCGGGTTTCCCTGAGCGAC GTATAAGAGACAG 266 hTRAV5 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCTCTGCGCATTGCAGACACCCA GTATAAGAGACAG 267 hTRAV6 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTGTTTCATATCACAGCCTCCCA GTATAAGAGACAG 268 hTRAV7 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GCTTGTACATTACAGCCGTGCA GTATAAGAGACAG 269 hTRAV8-1/8-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ATCTGAGGAAACCCTCTGTGCA GTATAAGAGACAG 270 hTRAV8-2/8-4 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACCTGACGAAACCCTCAGCCCAT GTATAAGAGACAG 271 hTRAV8-5 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTATGCCTGTCTTTACTTTAATC GTATAAGAGACAG 272 hTRAV8-6 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTGAGGAAACCCTCAGTCCATAT GTATAAGAGACAG 273 hTRAV8-7 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAAACCATCAACCCATGTGAGTGA GTATAAGAGACAG 274 hTRAV9-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACTTGGAGAAAGACTCAGTTCAA GTATAAGAGACAG 275 hTRAV9-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACTTGGAGAAAGGCTCAGTTCAA GTATAAGAGACAG 276 hTRAV10 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTGCACATCACAGCCTCCCA GTATAAGAGACAG 277 hTRAV11 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTTTGGAATATCGCAGCCTCTCAT GTATAAGAGACAG 278 hTRAV12-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTGCTCATCAGAGACTCCAAG GTATAAGAGACAG 279 hTRAV12-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTCTGCTCATCAGAGACTCCCAG GTATAAGAGACAG 280 hTRAV12-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTTGTTCATCAGAGACTCACAG GTATAAGAGACAG 281 hTRAV13-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCTGCACATCACAGAGACCCAA GTATAAGAGACAG 282 hTRAV13-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCTCTGCAAATTGCAGCTACTCAA GTATAAGAGACAG 283 hTRAV14 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTGTCATCTCCGCTTCACAACTGG GTATAAGAGACAG 284 hTRAV15 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTTTTGAATATGCTGGTCTCTCAT GTATAAGAGACAG 285 hTRAV16 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTGAAGAAACCATTTGCTCAAGA GTATAAGAGACAG 286 hTRAV17 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCTTGTTGATCACGGCTTCCCGG GTATAAGAGACAG 287 hTRAV18 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACCTGGAGAAGCCCTCGGTGCA GTATAAGAGACAG 288 hTRAV19 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CACCATCACAGCCTCACAAGTCGT GTATAAGAGACAG 289 hTRAV20 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTTCTGCACATCACAGCCCCTA GTATAAGAGACAG 290 hTRAV21 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTTATACATTGCAGCTTCTCAGCC GTATAAGAGACAG 291 hTRAV22 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTACATTTCCTCTTCCCAGACCAC GTATAAGAGACAG 292 hTRAV23 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CATTGCATATCATGGATTCCCAGC GTATAAGAGACAG 293 hTRAV24 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GCTATTTGTACATCAAAGGATCCC GTATAAGAGACAG 294 hTRAV25 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CAGCTCCCTGCACATCACAGCCA GTATAAGAGACAG 295 hTRAV26-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTGATCCTGCCCCACGCTACGCTGA GTATAAGAGACAG 296 hTRAV26-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTGATCCTGCACCGTGCTACCTTGA GTATAAGAGACAG 297 hTRAV27 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTTCTCTCCACATCACTGCAGCC GTATAAGAGACAG 298 hTRAV28 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GCCACCTATACATCAGATTCCCA GTATAAGAGACAG 299 hTRAV29 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCTCTGCACATTGTGCCCTCCCA GTATAAGAGACAG 300 hTRAV30 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTGTACCTTACGGCCTCCCAGCT GTATAAGAGACAG 301 hTRAV31 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTATCATATCATCATCACAGCCA GTATAAGAGACAG 302 hTRAV32 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCTGCATATTACAGCCACCCAA GTATAAGAGACAG 303 hTRAV33 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACCTCACCATCAATTCCTTAAAAC GTATAAGAGACAG 304 hTRAV34 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCTGCATATCACAGCCTCCCAG GTATAAGAGACAG 305 hTRAV35 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTCCTGAATATCTCAGCATCCAT GTATAAGAGACAG 306 hTRAV36 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCTGAACATCACAGCCACCCAG GTATAAGAGACAG 307 hTRAV37 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCTGCACATACAGGATTCCCAG GTATAAGAGACAG 308 hTRAV38 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CAAGATCTCAGACTCACAGCTGG GTATAAGAGACAG 309 hTRAV39 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCGTCTCAGCACCCTCCACATCA GTATAAGAGACAG 310 hTRAV40 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCATTGTGAAATATTCAGTCCAGG GTATAAGAGACAG

TABLE 10 Preferred primer sequences for amplification of TCR β chain V segments. N can be any nucleotide. The sequences for primers presented in this table consist of three parts (listed from 5′ to 3′): T7 adapter, barcode and TCR β chain V segment. SEQ Sequence T7 Sequence ID Primer adapter portion  barcode portion Sequence TCR β chain V segment NO name of the primer of the primer portion of the primer 311 hTRBV1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTGGTCGCACTGCAGCAAGAAGA GTATAAGAGACAG 312 hTRBV2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GATCCGGTCCACAAAGCTGGAGGA GTATAAGAGACAG 313 hTRBV3-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CATCAATTCCCTGGAGCTTGGTGA GTATAAGAGACAG 314 hTRBV4-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTCACCTACACGCCCTGCAGCCAG GTATAAGAGACAG 315 hTRBV4-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTCACCTACACACCCTGCAGCCAG GTATAAGAGACAG 316 hTRBV5-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAATGTGAGCACCTTGGAGCTGG GTATAAGAGACAG 317 hTRBV5-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TACTGAGTCAAACACGGAGCTAGG GTATAAGAGACAG 318 hTRBV5-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GCTCTGAGATGAATGTGAGTGCCT GTATAAGAGACAG 319 hTRBV5-4 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTGAGCTGAATGTGAACGCCTT GTATAAGAGACAG 320 hTRBV6-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGTTCTCGCTCAGGCTGGAGT GTATAAGAGACAG 321 hTRBV6-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTGGGGTTGGAGTCGGCTGCTC GTATAAGAGACAG 322 hTRBV6-4 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCCTCACGTTGGCGTCTGCTG GTATAAGAGACAG 323 hTRBV6-5 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCGCTCAGGCTGCTGTCGGC GTATAAGAGACAG 324 hTRBV6-6 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GATTTCCCGCTCAGGCTGGAGT GTATAAGAGACAG 325 hTRBV6-7 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCCCTCAAGCTGGAGTCAGCT GTATAAGAGACAG 326 hTRBV6-8 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCACTCAGGCTGGTGTCGGC GTATAAGAGACAG 327 hTRBV7-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTCTGAAGTTCCAGCGCACACA GTATAAGAGACAG 328 hTRBV7-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GATCCAGCGCACACAGCAGGAG GTATAAGAGACAG 329 hTRBV7-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACTCTGAAGATCCAGCGCACAGA GTATAAGAGACAG 330 hTRBV7-5 TCGTCGGCAGCGTCAGATGT HHHHHNNNN AGATCCAGCGCACAGAGCAAGG GTATAAGAGACAG 331 hTRBV7-6 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CAGCGCACAGAGCAGCGGGACT GTATAAGAGACAG 332 hTRBV7-9 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGATCCAGCGCACAGAGCAGG GTATAAGAGACAG 333 hTRBV8-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTCAACCCTGGAGTCTACTA GTATAAGAGACAG 334 hTRBV8-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCCAATCCTGGCATCCACCA GTATAAGAGACAG 335 hTRBV9 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTAAACCTGAGCTCTCTGGAGCT GTATAAGAGACAG 336 hTRBV10-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTCACTCTGGAGTCTGCTGC GTATAAGAGACAG 337 hTRBV10-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTCACTCTGGAGTCAGCTAC GTATAAGAGACAG 338 hTRBV10-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCTCACTCTGGAGTCCGCTAC GTATAAGAGACAG 339 hTRBV11-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCACTCTCAAGATCCAGCCTGCA GTATAAGAGACAG 340 hTRBV12-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGGATCCAGCCCATGGAACCCA GTATAAGAGACAG 341 hTRBV12-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTGAAGATCCAGCCTGCAGAGC GTATAAGAGACAG 342 hTRBV12-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CAGCCCTCAGAACCCAGGGACT GTATAAGAGACAG 343 hTRBV13 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGCTCCTTGGAGCTGGGGGACT GTATAAGAGACAG 344 hTRBV14 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGTGCAGCCTGCAGAACTGGAG GTATAAGAGACAG 345 hTRBV15 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GACATCCGCTCACCAGGCCTGG GTATAAGAGACAG 346 hTRBV16 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TGAGATCCAGGCTACGAAGCTT GTATAAGAGACAG 347 hTRBV17 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAAGATCCATCCCGCAGAGCCG GTATAAGAGACAG 348 hTRBV18 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGATCCAGCAGGTAGTGCGAGG GTATAAGAGACAG 349 hTRBV19 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CACTGTGACATCGGCCCAAAAG GTATAAGAGACAG 350 hTRBV20 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTGACAGTGACCAGTGCCCATC GTATAAGAGACAG 351 hTRBV21 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGATCCAGTCCACGGAGTCAG GTATAAGAGACAG 352 hTRBV22 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTGAAGTTGGCCCACACCAGCCA GTATAAGAGACAG 353 hTRBV23 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTGGCAATCCTGTCCTCAGAA GTATAAGAGACAG 354 hTRBV24 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGTCTGCCATCCCCAACCAGA GTATAAGAGACAG 355 hTRBV25 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGAGTCTGCCAGGCCCTCACA GTATAAGAGACAG 356 hTRBV26 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAAGTCTGCCAGCACCAACCAG GTATAAGAGACAG 357 hTRBV27 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGAGTCGCCCAGCCCCAACCAG GTATAAGAGACAG 358 hTRBV28 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGAGTCCGCCAGCACCAACCAG GTATAAGAGACAG 359 hTRBV29 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTGAGCAACATGAGCCCTGAAGA GTATAAGAGACAG 360 hTRBV30 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGTTCTAAGAAGCTCCTTCTCA GTATAAGAGACAG

TABLE 11 Primers for TCR gene amplification. Primer pair for sequencing of TCR  α genes: SEQ ID NOs: 256 and 257. Primer pair for sequencing of TCR β genes: SEQ ID NOs: 256 and 258. The primer portion corresponding to the  Illumina ® adapters (forward and reverse) is underlined in reverse primers shown below. SEQ ID NO Primer name Primer sequence TCR chain 256 Forward primer Nextera 5′ TCGTCGGCAGCGTC α and β 257 Reverse primer PCR 1 TRAV GTCTCGTGGGCTCGGAGATGTGTATAA GAGACAGGAATCAAAATCGGTGAATA α GGCAG 258 Reverse primer PCR 1 TRBV GTCTCGTGGGCTCGGAGATGTGTATAA GAGACAGGCCAGGCACACCAGTGTGG β CCTTTT

TABLE 12 Primers used to add the full Nextera sequence to both TCRα and TCRβ. SEQ ID TCR NO Primer name Primer sequence chain 259 Index Read 1 CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTC α and β 260 Index Read 2 AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCG α

Example 3: Exemplary Protocol for the SEQTR Method without In Vitro Transcription

TCR α and β chain genes were sequenced in two independent reactions.

1) Starting Material and RNA Extraction

-   -   To obtain sufficient amounts of RNA in the extraction, a minimum         of 500,000 T-cells were used as starting material.         Alternatively, and especially in instances where fewer T-cells         were available, T-cells were mixed with 50,000 mouse 3T3 cells         that served as carrier. T-cell RNA was extracted using the         RNeasy® Micro Kit from Qiagen Inc. according the manufacturer's         instruction with the following modification: Elution was         performed with 20 μl of water preheated to 50° C. RNA quality         and quantity was verified using a fragment analyzer.

2) cDNA Synthesis by Reverse Transcription:

-   -   The reverse transcription of the RNA was performed with the         SuperScript® III from Invitrogen (Thermo Fisher Scientific) and         oligo d(T). The kit provides the enzyme, the buffer and the         dithiothreitol (DTT) needed for the reaction. Deoxynucleotides         (dNTPs), oligo d(T) and RNAsin® Ribonuclease inhibitor were         purchased from Promega.     -   500 ng of RNA were used as starting material for the reverse         transcription. RNA was mixed with 1 μl of oligo d(T) and 1 μl         dNTP (25 mM) in a final volume of 13 μl. The mix was first         incubated at 70° C. for 10 min, then at 50° C. for 30 s. 4 μl 5×         buffer, 1 μl DTT (100 mM), 1 μl SuperScript III and 1 μl RNAsin®         were added to the mix. The samples were subsequently incubated         for at 55° C. 1 h and then at 85° C. for 5 min.

3) Second Strand cDNA Synthesis:

-   -   cDNA was then used to synthesize the second strand, performed         using the Phusion® High-Fidelity DNA polymerase (New England         Biolabs) under the following conditions:     -   Mix: 20 μl cDNA from step 2, 4 μl dNTPs (10 mM), 2 μl TRAV         primer mix (Table 9), 2 μl TRBV primers mix (Table 10), 20 μl 5×         buffer, 1 μl Phusion® enzyme in a total volume of 100 μl.     -   Synthesis conditions:         -   98° C. for 5 min         -   40° C. for 30 s         -   72° C. for 5 min

4) cDNA Purification:

-   -   100 μl of the cDNA product from step 3 were purified using an         AMPURE XP beads (Beckman Coulter) according to the         manufacturer's instruction.

5) TCR Gene Amplification:

-   -   TCR gene amplification was performed using a Phusion®         High-Fidelity DNA polymerase (New England Biolabs) under the         following conditions:     -   PCR mix: 7 μl cDNA from step 4, 0.4 μl dNTPs (10 mM), 0.4 μl         primer mix (20 μM Nextera5′, 10 μM Reverse primer PCR 1 TRAV or         2.5 μM Reverse primer PCR1 TRBV, see Table 11), 2 μl 5× buffer         and 0.2 μl Phusion® enzyme in a total volume of 10 μl.     -   PCR conditions:         -   94° C. for 5 min         -   20 cycles of             -   98° C. for 10 s             -   55° C. for 30 s             -   72° C. for 30 s         -   72° C. for 2 min     -   PCR products were purified using 1 μl of ExoSAP-IT® PCR Product         Cleanup Kit (Affymetrix) according to the manufacturer's         instructions.

6) Addition of Next Generation Sequencing Adapters:

-   -   Illumina® sequencing adapters were added by PCR using a Phusion®         High-Fidelity DNA polymerase (New England Biolabs). The         following mix was added to the 11 μl of PCR1: 1 μl dNTPs (10         mM), 1 μl primer mix (1.25 μM each, see Table 12), 3 μl 5×         buffer and 0.2 μl Phusion® enzyme and 9.8 μl of H₂O.     -   PCR conditions:         -   94° C. for 5 min         -   perform 25 cycles of:             -   98° C. for 10 s             -   55° C. for 30 s             -   72° C. for 30 s         -   72° C. for 2 min

7) TCR Library Purification:

-   -   10 μl of the PCR product from step 5 were purified using an         AMPURE XP beads (Beckman Coulter) according to the         manufacturer's instruction. Samples could then directly be used         for Illumina® sequencing.

Example 4: Sensitivity of the TCR Sequencing Method

One of the challenges of TCR sequencing are the small amounts of genetic material for each T-cell clone. In many cases, the number of T-cells that can be recovered from a given experiment is too small for researchers to directly extract sufficient amounts of RNA for a subsequent amplification of the TCR genes. In such instances, the T-cells of interest can be mixed with 3T3 mouse cells, which serve as a carrier.

5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively. The RNA of each mixture was isolated and subjected to steps 2 to 4 of the SEQTR method outlined above (see Detailed Description of the Invention). PCR products were separated on an agarose gel and visualized.

No TCR-specific PCR products were observed in samples that only contained 3T3 cells (see FIG. 4). However, TCR-specific bands were detected in all other samples: Increasing amounts of CD8 positive T-cells in the samples were correlated with increasing amounts of TCR-specific PCR products and decreasing intensity of the unspecific dimer primer band. These data demonstrate that the SEQTR method is sensitive enough to amplify TCR genes from as little as 1,000 T-cells, with no detectable background signal from the 3T3 carrier cells.

Example 5: Specificity of the SEQTR Method

Another challenge of TCR sequencing is the lack of specific amplification of TCR genes from complex samples. Competing TCR sequencing technologies such as services offered by Adaptive Biotechnology are characterized by up to 90% unspecific amplification. As a result, only as little as 10% of all sequencing data are informative for TCR repertoire determination, increasing cost and duration of any project aiming to sequence TCR repertoires.

5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively. TCR repertoires for the individual samples were sequenced using the SEQTR method, and the percentage of reads that corresponded to TCR or non-TCR sequences, respectively, was determined. As shown in FIG. 5, 93-97% of all sequencing reads indeed corresponded to TCR genes, independent of the amount of T-cells used as starting material. In summary, these data show that TCR amplification using the SEQTR method is highly specific even when as little as 1,000 T-cells are used as starting material.

Example 6: Unambiguous Identification of TCR Genes

In humans, the TCR locus comprises 54 different V segments for the TCR α chain and 65 different V segments for the TCR β chain. However, many of these V segments are highly homologous. Consequently, one of the big challenges of TCR sequencing is to successfully differentiate between two or more TCR gene segments with high degrees of homology. For instance, depending on the choice of primer used in the amplification of the TCR gene and the length of the generated PCR product, the resulting sequencing data might be compatible with more than one V or J segment (in other words, two or more TCR V or J segments show 100% homology in the sequenced region). In these cases, the TCR gene for a specific read cannot be unambiguously assigned/identified.

5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively. The RNA of each mixture was isolated and subjected to the TCR sequencing method. Out of all the sequencing reads that were identified as TCR genes, it was assessed if the V or J segments could be identified unambiguously. The data show that between 95% and 97% of all TCR sequencing reads could be assigned to a specific TCR segment, even when using as little as 1,000 T-cells as genetic starting material (see FIG. 6). In summary, the data demonstrate the robustness of the SEQTR method as 90 to 93% of all reads can be used to identify TCR sequences once unspecific sequences and ambiguous TCR sequences have been removed.

Due to the homology between V segments, it can be sometimes difficult to clearly identify the TCR sequence. hTRBV6-2 and hTRBV6-3 cannot be differentiated as they have 100% homology and thus will code for the same TCR. Due to their sequences, hTRBV12-3 and hTRBV12-4 cannot be differentiated with the method disclosed herein. Only paired-end sequencing that will catch the 5′-end of the V segment can discriminate these two sequences. Thus the hTRBV12-3 and hTRBV12-4 were considered as a unique sequence for the analysis of the repertoire.

Example 7: Linearity of TCR Gene Amplification

Because non-linear amplification of individual TCR sequences can lead to an incorrect over- or underrepresentation of the affected TCR genes in the final TCR repertoire, linearity of amplification is a critical determinant of the reliability and quality of the TCR sequencing data.

To test linearity of TCR gene amplification in our system, a fixed amount of DNA encoding a known TCR sequence was diluted at different concentrations into a DNA pool representing a naïve CD8 repertoire. Subsequently, the TCR repertoire of each sample was analyzed with SEQTR.

The observed frequency of the known TCR sequence in the entire TCR repertoire was then sequenced for each dilution and compared to the expected frequency. The scatter plot in FIG. 7 shows an excellent correlation (R²=0.99) between the dilution and the frequency of the known TCR sequence in the repertoire observed after sequencing. These data confirm the linearity of the amplification and suggest that results obtained using the SEQTR technique are quantitative.

Example 8: Reproducibility of the SEQTR Method

The reproducibility of the method was tested by performing two independent technical replicates starting from the same sample. The frequencies for each V-J rearrangement in the TCR β chains were determined and compared between the two replicates, as illustrated in FIG. 8. Each sphere represents a single V-J rearrangement that was detected in both replicates. Each sphere represents a single V-J rearrangement with the size of a sphere indicating the relative frequency of the specific V-J recombination. Grey spheres represent rearrangements for which the relative frequencies detected in the two replicates differed by less than two-fold. Black spheres represent rearrangements for which the relative frequencies detected in the two replicates differed by more than two-fold. Consistent with common practice in the analysis of gene expression data, differences between replaces of less than 2-fold are not considered significant.

The data show that only 13% of all V-J rearrangements showed a significant frequency difference of more than two-fold between the two technical replicates (see FIG. 8 upper inset). However, as illustrated in FIG. 8, V-J recombinations that were significantly different between the technical replicates were rather poorly expressed, as indicated by the small sizes of the black spheres. Therefore, if the frequencies of the individual V-J rearrangement are taken into consideration, only 0.5% of the sequences showed more than a two-fold difference between the replicates (see FIG. 8 lower inset), demonstrating that the SEQTR method is very reproducible.

Example 9: Sequencing of Example Repertoires Using the SEQTR Method

The SEQTR method was tested on three different type of CD8 positive T-cells:

-   -   (1) T-cell population 1: CD8 positive T-cells isolated from         peripheral blood mononuclear cells (PBMCs).     -   (2) T-cell population 2: CD8 positive T-cells as in population 1         were FACS sorted using tetramers. Tetramers are MHC molecules         presenting a specific peptide, linked to fluorescent dye.         Tetramers bind T-cell expressing a TCR that specifically         recognizes the peptide. The fluorescent dye allows sorting of         the the desired T-cells by FACS.     -   (3) T-cell population 3: CD8 positive T-cells as in population 2         that were subsequently expanded in vitro.

The relative frequencies of each V-J rearrangement were determined using the SEQTR method (see FIG. 9). As expected, the naïve TCR repertoire derived from PBMC (population 1) is highly diverse (see FIG. 9A). Almost all the possible V-J rearrangements are represented in the sample, with no single V-J rearrangement exhibiting a frequency of over 11% The repertoire of the tetramer sorted CD8 positive T-cell subset 2 (see FIG. 9B) is less diverse as compared to the naïve one. Not only are fewer V-J rearrangements present in the repertoire overall. Moreover, two V-J rearrangements are clearly dominant, exhibiting frequencies of over 20%. These V-J rearrangements represent the few T-cells that recognize the epitope TEDYMIHII (SEQ ID NO: 236) conjugated to the tetramer and that were enriched during the tetramer purification step. Finally, the rapid clonal expansion (population 3) of the tetramer-purified T-cells enhances the bias of the TCR repertoire towards the T-cell clones already dominating subset 2. Consequently, part of the low frequency V-J rearrangements are lost and not detected anymore (see FIG. 9C). In summary, these data illustrate that the SEQTR method is well suited to differentiate between TCR repertoires with different degrees of diversities.

Example 10: Comparison of the SEQTR Method with Low-Throughput Single Cell Cloning

In order to determine how accurate the TCR repertoire data obtained using the SEQTR method were as compared to the true TCR repertoire present in a given T-cell population, we compared our results to data obtained by single cell sequencing.

Tetramer-specific CD8 were sorted from PBMC by FACS. The recovered cell population was split in two. Half of the cells were subjected to the SEQTR method to sequence the TCR repertoire. For the other half of the cells, individual T-cell clones were isolated and expanded in vitro (single cell cloning). Once the clones were established, the TCR genes of each T-cell clones were amplified and sequenced using classical Sanger sequencing (see FIG. 10A).

Among the 42 individual clones tested using the single cell method, six different TCRs were identified (see FIG. 10B). Using the SEQTR method, 116 different TCR genes were found (the eight most frequently observed V-J rearrangements are shown in FIG. 10C, also see Table 13 and Table 14). Indeed, the five TCRs most frequently observed with the single cell cloning technique also correspond to the five clones most frequently observed when applying the SEQTR method. Overall, all six TCR clones identified with single cell sequencing are represented among the eight TCRs with the highest frequencies observed in the SEQTR method. In summary, these data suggest that the SEQTR method produces a true representation of the actual TCR repertoire of a given T-cell population.

TABLE 13 CDR3 regions of TCR clones identified using the single cell sequencing method. SEQ ID NO CDR3 region 237 CASSRHVGGVPEAFFG 238 CASSIGRGSEQYFG 239 CASSDVLSGEAFFG 240 CASQGHKNTEAFFG 241 CASSLGPGGVKTNEKLFFG 242 CASSLGPGGVKTNEKLFFG

TABLE 14 Eight most frequently observed V-J rearrangements of the 116 different TCR genes identified using the SEQTR method. SEQ ID NO CDR3 region 243 CASSDVLSGEAFFG 244 CASQGHKNTEAFFG 245 CASSLGPGGVKTNEKLFFG 246 CASSIGRGSEQYFG 247 CASSRHVGGVPEAFFG 248 CASSASKGQPQHFG 249 CASQGHKNTEAFFG 250 CASSLGPGGVKTNEKLFFG

Example 11: TCR Sequencing Services Offered by Adaptive Biotechnology Provide Sequencing Data that May Reflect Up to 90% Unspecific TCR Amplification

Tumor samples from 16 patients were collected and the tumor cells were separated from the surrounding tissue (stroma). In addition, epitope-specific TIL were sorted by FACS from the tumor samples using tetramer staining (TET). Finally, the tumor cells were engrafted into humanized mice. After some time, the tumor was collected and epitope specific TIL were sorted by FACS.

DNA extraction was performed for each sample. DNA was sent to Adaptive Biotechnology for TCR sequencing (immunoSEQ® method, survey protocol 200,000-300,000 reads per sample).

In 80% of the samples, the immunoSEQ® method failed to generate 200,000 reads per samples, suggesting that the immunoSEQ® method fails to generate TCR repertoires with significant reliability.

Example 12: Amplification of TCR Genes from PBMC and CD4 Positive T-Cells

RNA was isolated from 10{circumflex over ( )}6 PBMC or 10{circumflex over ( )}6 CD4 positive T-cells, respectively, from three independent samples, The RNA was then subjected to steps 2 to 4 of the SEQTR method outlined above (see Detailed Description of the Invention). PCR products were separated on an agarose gel and visualized (see FIG. 12). Only TCR-specific bands are observed, suggesting that the SEQTR method cannot only be used for CD8 positive T-cells (see Example 7), but also for CD4 positive T-cells and even for T-cells that are part of a complex mixture of other PBMCs.

The foregoing examples and description of the embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference herein in their entireties.

As described and claimed herein, including in the accompanying drawings, reference is made to particular features, including method steps. It is to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment, or a particular claim, that feature can also be used, to the extent possible, in combination with and/or in the context of other particular aspects and embodiments, and in the disclosed methods, systems and kits generally.

Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes that possibility).

TABLE 15 TCR α chain V segments and binding sites for primers presented in Table 2 and Table 9. The sequence for each V segment presented in this table consists of three parts (listed from 5′ to 3′): Sequence upstream of primer binding site, sequence of the primer binding site, sequence downstream of the primer binding site. V SEQ Primer hTRAV sequence segment Primer ID binding site downstream of name name NO hTRAV sequence upstream of primer binding site within hTRAV primer binding site hTRAV01-1 hTRAV01-1 117 ATGTGGGGAGCTTTCCTTCTCTATGTTTCCATGAAGATGGGAGGCACTGCAGGACAAAGCCTTGAGCAGCCCTCT CTTCTACAG ACTCTGCCTCTTA GAAGTGACAGCTGTGGAAGGAGCCATTGTCCAGATAAACTGCACGTACCAGACATCTGGGTTTTATGGGCTGTC GAGCTCCAG CTTCTGCGCTGT CTGGTACCAGCAACATGATGGCGGAGCACCCACATTTCTTTCTTACAATGCTCTGGATGGTTTGGAGGAGACAGG ATGAAAG GAGAGA TCGTTTTTCTTCATTCCTTAGTCGCTCTGATAGTTATGGTTACCTC hTRAV01-2 hTRAV01-2 118 ATGTGGGGAGTTTTCCTTCTTTATGTTTCCATGAAGATGGGAGGCACTACAGGACAAAACATTGACCAGCCCACT CTTTTGAAG ACTCTGCCTCTTA GAGATGACAGCTACGGAAGGTGCCATTGTCCAGATCAACTGCACGTACCAGACATCTGGGTTCAACGGGCTGTT GAGCTCCAG CCTCTGTGCTGT CTGGTACCAGCAACATGCTGGCGAAGCACCCACATTTCTGTCTTACAATGTTCTGGATGGTTTGGAGGAGAAAGG ATGAAAG GAGAGA TCGTTTTTCTTCATTCCTTAGTCGGTCTAAAGGGTACAGTTACCTC hTRAV02 hTRAV02 119 ATGGCTTTGCAGAGCACTCTGGGGGCGGTGTGGCTAGGGCTTCTCCTCAACTCTCTCTGGAAGGTTGCAGAAAGC TGCTCATCC GGCAGATGCTGC AAGGACCAAGTGTTTCAGCCTTCCACAGTGGCATCTTCAGAGGGAGCTGTGGTGGAAATCTTCTGTAATCACTCT TCCAGGTGC TGTTTACTACTGT GTGTCCAATGCTTACAACTTCTTCTGGTACCTTCACTTCCCGGGATGTGCACCAAGACTCCTTGTTAAAGGCTCAA GGGA GCTGTGGAGGA AGCCTTCTCAGCAGGGACGATACAACATGACCTATGAACGGTTCTCTTCATCGC hTRAV03 hTRAV03 120 ATGGCCTCTGCACCCATCTCGATGCTTGCGATGCTCTTCACATTGAGTGGGCTGAGAGCTCAGTCAGTGGCTCAG GAAGAAACC GCGACTCCGCTT CCGGAAGATCAGGTCAACGTTGCTGAAGGGAATCCTCTGACTGTGAAATGCACCTATTCAGTCTCTGGAAACCCT ATCTGCCCT TGTACTTCTGTGC TATCTTTTTTGGTATGTTCAATACCCCAACCGAGGCCTCCAGTTCCTTCTGAAATACATCACAGGGGATAACCTGG TGTGA TGTGAGAGACA TTAAAGGCAGCTATGGCTTTGAAGCTGAATTTAACAAGAGCCAAACCTCCTTCCACCT hTRAV04 hTRAV04 121 ATGAGGCAAGTGGCGAGAGTGATCGTGTTCCTGACCCTGAGTACTTTGAGCCTTGCTAAGACCACCCAGCCCATC CCTGCCCCG ACTGCTGTGTAC TCCATGGACTCATATGAAGGACAAGAAGTGAACATAACCTGTAGCCACAACAACATTGCTACAAATGATTATAT GGTTTCCCT TACTGCCTCGTG CACGTGGTACCAACAGTTTCCCAGCCAAGGACCACGATTTATTATTCAAGGATACAAGACAAAAGTTACAAACG GAGCGAC GGTGACA AAGTGGCCTCCCTGTTTATCCCTGCCGACAGAAAGTCCAGCACTCTGAG hTRAV05 hTRAV05 122 ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCTGGACTGTATGAGTAGAGGAGAGGATGTG TCTCTGCGC GACTGGGGACTC GAGCAGAGTCTTTTCCTGAGTGTCCGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGCTCCTCC ATTGCAGAC AGCTATCTACTT ACCTACTTATACTGGTATAAGCAAGAACCTGGAGCAGGTCTCCAGTTGCTGACGTATATTTTTTCAAATATGGAC ACCCA CTGTGCAGAGAG ATGAAACAAGACCAAAGACTCACTGTTCTATTGAATAAAAAGGATAAACATCTG TA hTRAV06 hTRAV06 123 ATGGAGTCATTCCTGGGAGGTGTTTTGCTGATTTTGTGGCTTCAAGTGGACTGGGTGAAGAGCCAAAAGATAGAA TTGTTTCAT GCCTGCAGACTC CAGAATTCCGAGGCCCTGAACATTCAGGAGGGTAAAACGGCCACCCTGACCTGCAACTATACAAACTATTCCCC ATCACAGCC AGCTACCTACCT AGCATACTTACAGTGGTACCGACAAGATCCAGGAAGAGGCCCTGTTTTCTTGCTACTCATACGTGAAAATGAGA TCCCA CTGTGCTCTAGA AAGAAAAAAGGAAAGAAAGACTGAAGGTCACCTTTGATACCACCCTTAAACAGAGT CA hTRAV07 hTRAV07 124 ATGGAGAAGATGCGGAGACCTGTCCTAATTATATTTTGTCTATGTCTTGGCTGGGCAAATGGAGAAAACCAGGTG GCTTGTACA GCCTGAAGATTC GAGCACAGCCCTCATTTTCTGGGACCCCAGCAGGGAGACGTTGCCTCCATGAGCTGCACGTACTCTGTCAGTCGT TTACAGCCG AGCCACCTATTT TTTAACAATTTGCAGTGGTACAGGCAAAATACAGGGATGGGTCCCAAACACCTATTATCCATGTATTCAGCTGGA TGCA CTGTGCTGTAGA TATGAGAAGCAGAAAGGAAGACTAAATGCTACATTACTGAAGAATGGAAGCA TG hTRAV08-1 hTRAV08- 125 ATGCTCCTGTTGCTCATACCAGTGCTGGGGATGATTTTTGCCCTGAGAGATGCCAGAGCCCAGTCTGTGAGCCAG ATCTGAGGA GTGGAGTGACAC 1/08-3 CATAACCACCACGTAATTCTCTCTGAAGCAGCCTCACTGGAGTTGGGATGCAACTATTCCTATGGTGGAACTGTT AACCCTCTG AGCTGAGTACTT AATCTCTTCTGGTATGTCCAGTACCCTGGTCAACACCTTCAGCTTCTCCTCAAGTACTTTTCAGGGGATCCACTGG TGCA CTGTGCCGTGAA TTAAAGGCATCAAGGGCTTTGAGGCTGAATTTATAAAGAGTAAATTCTCCTTTA TGC hTRAV08-2 hTRAV08- 126 ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACTCTGGGAGGAACCAGAGCCCAGTCGGTGACCCAG ACCTGACGA ATGAGCGACGCG 2/08-4 CTTGACAGCCACGTCTCTGTCTCTGAAGGAACCCCGGTGCTGCTGAGGTGCAACTACTCATCTTCTTATTCACCAT AACCCTCAG GCTGAGTACTTC CTCTCTTCTGGTATGTGCAACACCCCAACAAAGGACTCCAGCTTCTCCTGAAGTACACATCAGCGGCCACCCTGG CCCAT TGTGTTGTGAGT TTAAAGGCATCAACGGTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCC GA hTRAV08-3 hTRAV08- 127 ATGCTCCTGGAGCTTATCCCACTGCTGGGGATACATTTTGTCCTGAGAACTGCCAGAGCCCAGTCAGTGACCCAG ATCTGAGGA TTGGAGTGATGC 1/08-3 CCTGACATCCACATCACTGTCTCTGAAGGAGCCTCACTGGAGTTGAGATGTAACTATTCCTATGGGGCAACACCT AACCCTCTG TGCTGAGTACTT TATCTCTTCTGGTATGTCCAGTCCCCCGGCCAAGGCCTCCAGCTGCTCCTGAAGTACTTTTCAGGAGACACTCTGG TGCA CTGTGCTGTGGG TTCAAGGCATTAAAGGCTTTGAGGCTGAATTTAAGAGGAGTCAATCTTCCTTCA TGC hTRAV08-4 hTRAV08- 128 ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACCCTGGGAGGAACCAGAGCCCAGTCGGTGACCCAG ACCTGACGA ATGAGCGACGCG 2/08-4 CTTGGCAGCCACGTCTCTGTCTCTGAAGGAGCCCTGGTTCTGCTGAGGTGCAACTACTCATCGTCTGTTCCACCAT AACCCTCAG GCTGAGTACTTC ATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTACACATCAGCGGCCACCCTGG CCCAT TGTGCTGTGAGT TTAAAGGCATCAACGGTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCC GACACA hTRAV08-5 hTRAV08-5 129 ATGCTCCTGGTGCTCATCCCACTGCTGGGGATACATTTTGTCCTGAGTGAGAACTGTCAGAGCCCAGTCAGTGAC CCTATGCCT TCTTAATCCTGTC CCAGCCTGACATCCGCATCACTGTCTCTGAAGGAGCCTCACTGGAGTTGAGATGTAACTATTCCTATGGGGCGAT GTCTTTACT AGCTGAGGAGGA GTTGTGGGAAGTCAGGGACCCCAAACGGAGGGACCGGCTGAAGCCATGGCAGAAGAATGTGGATTGTGAAGAT TTAATC TGTATGTCACC TTCATGGACATTTATTAGTTCCCCAAATTAATACTTTTATAATTTCTTATGCCTCTCTTTACTGCAATCTCTAAACA TAAATTGTAAAGATTTCATGGACACTTATCACTTCCCCAATCAATACCCCTGTGATTT hTRAV08-6 hTRAV08-6 130 ATGCTCCTGCTGCTCGTCCCAGCGTTCCAGGTGATTTTTACCCTGGGAGGAACCAGAGCCCAGTCTGTGACCCAG CTTGAGGAA AAGCGACACGGC CTTGACAGCCAAGTCCCTGTCTTTGAAGAAGCCCCTGTGGAGCTGAGGTGCAACTACTCATCGTCTGTTTCAGTG ACCCTCAGT TGAGTACTTCTG TATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTATTTATCAGGATCCACCCTGG CCATAT TGCTGTGAGTGA TTAAAGGCATCAACGGTTTTGAGGCTGAATTTAACAAGAGTCAAACTTCCTTCCA hTRAV08-7 hTRAV08-7 131 ATGCTCTTAGTGGTCATTCTGCTGCTTGGAATGTTCTTCACACTGAGAACCAGAACCCAGTCGGTGACCCAGCTT GAAACCATC TGCTGCTGAGTA GATGGCCACATCACTGTCTCTGAAGAAGCCCCTCTGGAACTGAAGTGCAACTATTCCTATAGTGGAGTTCCTTCT AACCCATGT CTTCTGTGCTGTG CTCTTCTGGTATGTCCAATACTCTAGCCAAAGCCTCCAGCTTCTCCTCAAAGACCTAACAGAGGCCACCCAGGTT GAGTGA GGTGACAGG AAAGGCATCAGAGGTTTTGAGGCTGAATTTAAGAAGAGCGAAACCTCCTTCTACCTGAG hTRAV09-1 hTRAV09-1 132 ATGAATTCTTCTCCAGGACCAGCGATTGCACTATTCTTAATGTTTGGGGGAATCAATGGAGATTCAGTGGTCCAG ACTTGGAGA GAGTCAGACTCC ACAGAAGGCCAAGTGCTCCCCTCTGAAGGGGATTCCCTGATTGTGAACTGCTCCTATGAAACCACACAGTACCCT AAGACTCAG GCTGTGTACTTCT TCCCTTTTTTGGTATGTCCAATATCCTGGAGAAGGTCCACAGCTCCACCTGAAAGCCATGAAGGCCAATGACAAG TTCAA GTGCTCTGAGTG GGAAGGAACAAAGGTTTTGAAGCCATGTACCGTAAAGAAACCACTTCTTTCC A hTRAV09-2 hTRAV09-2 133 ATGAACTATTCTCCAGGCTTAGTATCTCTGATACTCTTACTGCTTGGAAGAACCCGTGGAAATTCAGTGACCCAG ACTTGGAGA GTGTCAGACTCA ATGGAAGGGCCAGTGACTCTCTCAGAAGAGGCCTTCCTGACTATAAACTGCACGTACACAGCCACAGGATACCC AAGGCTCAG GCGGTGTACTTC TTCCCTTTTCTGGTATGTCCAATATCCTGGAGAAGGTCTACAGCTCCTCCTGAAAGCCACGAAGGCTGATGACAA TTCAA TGTGCTCTGAGT GGGAAGCAACAAAGGTTTTGAAGCCACATACCGTAAAGAAACCACTTCTTTCC GA hTRAV10 hTRAV10 134 ATGAAAAAGCATCTGACGACCTTCTTGGTGATTTTGTGGCTTTATTTTTATAGGGGGAATGGCAAAAACCAAGTG CTGCACATC GCTCAGCGATTC GAGCAGAGTCCTCAGTCCCTGATCATCCTGGAGGGAAAGAACTGCACTCTTCAATGCAATTATACAGTGAGCCCC ACAGCCTCC AGCCTCCTACAT TTCAGCAACTTAAGGTGGTATAAGCAAGATACTGGGAGAGGTCCTGTTTCCCTGACAATCATGACTTTCAGTGAG CA CTGTGTGGTGAG AACACAAAGTCGAACGGAAGATATACAGCAACTCTGGATGCAGACACAAAGCAAAGCTCT CG hTRAV11 hTRAV11 135 ACGGAGAAGCCCTTGGGAGTTTCATTCTTGATTTCCTCCTGGCAGCTGTGCTGGGTGAATAGACTACATACACTG GTTTGGAAT CTGGGAGATTCA GAGCAGAGTCCTTCATTCCTGAATATTCAGGAGGGAATGCATGCCGTTCTTAATTGTACTTATCAGGAGAGAACA ATCGCAGCC GCCACCTACTTC CTCTTCAATTTCCACTGGTTCCGGCAGGATCCGGGGAGAAGACTTGTGTCTTTGACCTTAATTCAATCAAGCCAG TCTCAT TGTGCTTTGC AAGGAGCAGGGAGACAAATATTTTAAAGAACTGCTTGGAAAAGAAAAATTTTATAGT hTRAV12-1 hTRAV12-1 136 ATGATATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTGGGTTTGGAGCCAACGGAAGGAGGTG CCCTGCTCA CTCAGTGATTCA GAGCAGGATCCTGGACCCTTCAATGTTCCAGAGGGAGCCACTGTCGCTTTCAACTGTACTTACAGCAACAGTGCT TCAGAGACT GCCACCTACCTC TCTCAGTCTTTCTTCTGGTACAGACAGGATTGCAGGAAAGAACCTAAGTTGCTGATGTCCGTATACTCCAGTGGT CCAAG TGTGTGGTGAAC AATGAAGATGGAAGGTTTACAGCACAGCTCAATAGAGCCAGCCAGTATATTT A hTRAV12-2 hTRAV12-2 137 ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTG CTCTGCTCA CCCAGTGATTCA GAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGT TCAGAGACT GCCACCTACCTC TCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGT CCCAG TGTGCCGTGAA GACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTT hTRAV12-3 hTRAV12-3 138 ATGAAATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTGGGTTTGGAGCCAACAGAAGGAGGTG CCTTGTTCA CCCAGTGATTCA GAGCAGGATCCTGGACCACTCAGTGTTCCAGAGGGAGCCATTGTTTCTCTCAACTGCACTTACAGCAACAGTGCT TCAGAGACT GCCACCTACCTC TTTCAATACTTCATGTGGTACAGACAGTATTCCAGAAAAGGCCCTGAGTTGCTGATGTACACATACTCCAGTGGT CACAG TGTGCAATGAGC AACAAAGAAGATGGAAGGTTTACAGCACAGGTCGATAAATCCAGCAAGTATATCT GCACAG hTRAV13-1 hTRAV13-1 139 ATGACATCCATTCGAGCTGTATTTATATTCCTGTGGCTGCAGCTGGACTTGGTGAATGGAGAGAATGTGGAGCAG TCCCTGCAC CCTGAAGACTCG CATCCTTCAACCCTGAGTGTCCAGGAGGGAGACAGCGCTGTTATCAAGTGTACTTATTCAGACAGTGCCTCAAAC ATCACAGAG GCTGTCTACTTCT TACTTCCCTTGGTATAAGCAAGAACTTGGAAAAGGACCTCAGCTTATTATAGACATTCGTTCAAATGTGGGCGAA ACCCAA GTGCAGCAAGTA AAGAAAGACCAACGAATTGCTGTTACATTGAACAAGACAGCCAAACATTTC hTRAV13-2 hTRAV13-2 140 ATGGCAGGCATTCGAGCTTTATTTATGTACTTGTGGCTGCAGCTGGACTGGGTGAGCAGAGGAGAGAGTGTGGG TCTCTGCAA CCTGGAGACTCA GCTGCATCTTCCTACCCTGAGTGTCCAGGAGGGTGACAACTCTATTATCAACTGTGCTTATTCAAACAGCGCCTC ATTGCAGCT GCTGTCTACTTTT AGACTACTTCATTTGGTACAAGCAAGAATCTGGAAAAGGTCCTCAATTCATTATAGACATTCGTTCAAATATGGA ACTCAA GTGCAGAGAATA CAAAAGGCAAGGCCAAAGAGTCACCGTTTTATTGAATAAGACAGTGAAACATCTC hTRAV14 hTRAV14 141 ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGACCTGGCATTGCCCAGAAGATAACT TTGTCATCT GGGACTCAGCAA CAAACCCAACCAGGAATGTTCGTGCAGGAAAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGTGATCA CCGCTTCAC TGTATTTCTGTGC AAGTTATGGTCTATTCTGGTACAAGCAGCCCAGCAGTGGGGAAATGATTTTTCTTATTTATCAGGGGTCTTATGA AACTGG AATGAGAGAGGG CGAGCAAAATGCAACAGAAGGTCGCTACTCATTGAATTTCCAGAAGGCAAGAAAATCCGCCAACC hTRAV15 hTRAV15 142 ATGTATACGTATGTAACAAACCTGCGCGTTGTGCACATGTACCCTAGAACGGGTGAACAGCCTCCATATTCTGGA GTTTTGAAT CCTGGAGATTCA GTAGAGTCCTTCATTCATTCCTGAGTATCCGGGAGGGAATGCACAACATTCTTAATTGCACTTATGAGGAGAGAA ATGCTGGTC GGCACCTACTTC CGTTCTCTTAACTTCTACTGGTTCTGGCAGGGTCTGGAAAAGGACTTGTGTCTTTGACCTTAATTCAATCAAGCCA TCTCAT TGTGCTTTGAGG GATGGAGGAGGGAGACAAACATTTTAAAGAAGCGCTTGGAAAAGAGAAGTTTTATAGT hTRAV16 hTRAV16 143 ATGAAGCCCACCCTCATCTCAGTGCTTGTGATAATATTTATACTCAGAGGAACAAGAGCCCAGAGAGTGACTCA CCTGAAGAA GGAAGACTCAGC GCCCGAGAAGCTCCTCTCTGTCTTTAAAGGGGCCCCAGTGGAGCTGAAGTGCAACTATTCCTATTCTGGGAGTCC ACCATTTGC CATGTATTACTG TGAACTCTTCTGGTATGTCCAGTACTCCAGACAACGCCTCCAGTTACTCTTGAGACACATCTCTAGAGAGAGCAT TCAAGA TGCTCTAAGTGG CAAAGGCTTCACTGCTGACCTTAACAAAGGCGAGACATCTTTCCA hTRAV17 hTRAV17 144 ATGGAAACTCTCCTGGGAGTGTCTTTGGTGATTCTATGGCTTCAACTGGCTAGGGTGAACAGTCAACAGGGAGAA TCCTTGTTG GCAGCAGACACT GAGGATCCTCAGGCCTTGAGCATCCAGGAGGGTGAAAATGCCACCATGAACTGCAGTTACAAAACTAGTATAAA ATCACGGCT GCTTCTTACTTCT CAATTTACAGTGGTATAGACAAAATTCAGGTAGAGGCCTTGTCCACCTAATTTTAATACGTTCAAATGAAAGAGA TCCCGG GTGCTACGGACG GAAACACAGTGGAAGATTAAGAGTCACGCTTGACACTTCCAAGAAAAGCAGT hTRAV18 hTRAV18 145 ATGCTGTCTGCTTCCTGCTCAGGACTTGTGATCTTGTTGATATTCAGAAGGACCAGTGGAGACTCGGTTACCCAG ACCTGGAGA GCTGTCGGACTC ACAGAAGGCCCAGTTACCCTCCCTGAGAGGGCAGCTCTGACATTAAACTGCACTTATCAGTCCAGCTATTCAACT AGCCCTCGG TGCCGTGTACTA TTTCTATTCTGGTATGTCCAGTATCTAAACAAAGAGCCTGAGCTCCTCCTGAAAAGTTCAGAAAACCAGGAGACG TGCA CTGCGCTCTGAG GACAGCAGAGGTTTTCAGGCCAGTCCTATCAAGAGTGACAGTTCCTTCC A hTRAV19 hTRAV19 146 ATGCTGACTGCCAGCCTGTTGAGGGCAGTCATAGCCTCCATCTGTGTTGTATCCAGCATGGCTCAGAAGGTAACT CACCATCAC GGACTCAGCAGT CAAGCGCAGACTGAAATTTCTGTGGTGGAGAAGGAGGATGTGACCTTGGACTGTGTGTATGAAACCCGTGATAC AGCCTCACA ATACTTCTGTGCT TACTTATTACTTATTCTGGTACAAGCAACCACCAAGTGGAGAATTGGTTTTCCTTATTCGTCGGAACTCTTTTGAT AGTCGT CTGAGTGAGGC GAGCAAAATGAAATAAGTGGTCGGTATTCTTGGAACTTCCAGAAATCCACCAGTTCCTTCAACTT hTRAV20 hTRAV20 147 ATGGAGAAAATGTTGGAGTGTGCATTCATAGTCTTGTGGCTTCAGCTTGGCTGGTTGAGTGGAGAAGACCAGGTG TTTCTGCAC AACCTGAAGACT ACGCAGAGTCCCGAGGCCCTGAGACTCCAGGAGGGAGAGAGTAGCAGTCTTAACTGCAGTTACACAGTCAGCGG ATCACAGCC CAGCCACTTATC TTTAAGAGGGCTGTTCTGGTATAGGCAAGATCCTGGGAAAGGCCCTGAATTCCTCTTCACCCTGTATTCAGCTGG CCTA TCTGTGCTGTGC GGAAGAAAAGGAGAAAGAAAGGCTAAAAGCCACATTAACAAAGAAGGAAAGC AGG hTRAV21 hTRAV21 148 ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGAGCAGCAAACAGGAGGTGACGCA CTTTATACA TGGTGACTCAGC GATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCTATTTA TTGCAGCTT CACCTACCTCTG CAACCTCCAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTCACATCTCTGTTGCTTATTCAGTCAAGTCAGAGAGA CTCAGCC TGCTGTGAGG GCAAACAAGTGGAAGACTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTA hTRAV22 hTRAV22 149 ATGAAGAGGATATTGGGAGCTCTGCTGGGGCTCTTGAGTGCCCAGGTTTGCTGTGTGAGAGGAATACAAGTGGA GTACATTTC AGACTCAGGCGT GCAGAGTCCTCCAGACCTGATTCTCCAGGAGGGAGCCAATTCCACGCTGCGGTGCAATTTTTCTGACTCTGTGAA CTCTTCCCA TTATTTCTGTGCT CAATTTGCAGTGGTTTCATCAAAACCCTTGGGGACAGCTCATCAACCTGTTTTACATTCCCTCAGGGACAAAACA GACCAC GTGGAGC GAATGGAAGATTAAGCGCCACGACTGTCGCTACGGAACGCTACAGCTTATT hTRAV23 hTRAV23 150 ATGGACAAGATCTTAGGAGCATCATTTTTAGTTCTGTGGCTTCAACTATGCTGGGTGAGTGGCCAACAGAAGGAG CATTGCATA CTGGAGACTCAG AAAAGTGACCAGCAGCAGGTGAAACAAAGTCCTCAATCTTTGATAGTCCAGAAAGGAGGGATTTCAATTATAAA TCATGGATT CCACCTACTTCT CTGTGCTTATGAGAACACTGCGTTTGACTACTTTCCATGGTACCAACAATTCCCTGGGAAAGGCCCTGCATTATT CCCAGC GTGCAGCAAGCA GATAGCCATACGTCCAGATGTGAGTGAAAAGAAAGAAGGAAGATTCACAATCTCCTTCAATAAAAGTGCCAAGC AGTTCT hTRAV24 hTRAV24 151 ATGGAGAAGAATCCTTTGGCAGCCCCATTACTAATCCTCTGGTTTCATCTTGACTGCGTGAGCAGCATACTGAAC GCTATTTGT AGCCTGAAGACT GTGGAACAAAGTCCTCAGTCACTGCATGTTCAGGAGGGAGACAGCACCAATTTCACCTGCAGCTTCCCTTCCAGC ACATCAAAG CAGCCACATACC AATTTTTATGCCTTACACTGGTACAGATGGGAAACTGCAAAAAGCCCCGAGGCCTTGTTTGTAATGACTTTAAAT GATCCC TCTGTGCCTTTA GGGGATGAAAAGAAGAAAGGACGAATAAGTGCCACTCTTAATACCAAGGAGGGTTACA hTRAV25 hTRAV25 152 ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAGGTGAATGGACAACAGGTAATGCAAATT CAGCTCCCT CCCAGACTACAG CCTCAGTACCAGCATGTACAAGAAGGAGAGGACTTCACCACGTACTGCAATTCCTCAACTACTTTAAGCAATATA GCACATCAC ATGTAGGAACCT CAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTGATACAGTTAGTGAAGAGTGGAGAAGTGAAGAA AGCCA ACTTCTGTGCAG GCAGAAAAGACTGACATTTCAGTTTGGAGAAGCAAAAAAGAA GG hTRAV26-1 hTRAV26-1 153 ATGAGGCTGGTGGCAAGAGTAACTGTGTTTCTGACCTTTGGAACTATAATTGATGCTAAGACCACCCAGCCCCCC TTGATCCTG GAGACACTGCTG TCCATGGATTGCGCTGAAGGAAGAGCTGCAAACCTGCCTTGTAATCACTCTACCATCAGTGGAAATGAGTATGTG CCCCACGCT TGTACTATTGCA TATTGGTATCGACAGATTCACTCCCAGGGGCCACAGTATATCATTCATGGTCTAAAAAACAATGAAACCAATGA ACGCTGA TCGTCAGAGTCG AATGGCCTCTCTGATCATCACAGAAGACAGAAAGTCCAGCACC hTRAV26-2 hTRAV26-2 154 ATGAAGTTGGTGACAAGCATTACTGTACTCCTATCTTTGGGTATTATGGGTGATGCTAAGACCACACAGCCAAAT TTGATCCTG GAGATGCTGCTG TCAATGGAGAGTAACGAAGAAGAGCCTGTTCACTTGCCTTGTAACCACTCCACAATCAGTGGAACTGATTACATA CACCGTGCT TGTACTACTGCA CATTGGTATCGACAGCTTCCCTCCCAGGGTCCAGAGTACGTGATTCATGGTCTTACAAGCAATGTGAACAACAGA ACCTTGA TCCTGAGAGAC ATGGCCTCTCTGGCAATCGCTGAAGACAGAAAGTCCAGTACC hTRAV27 hTRAV27 155 ATGGTCCTGAAATTCTCCGTGTCCATTCTTTGGATTCAGTTGGCATGGGTGAGCACCCAGCTGCTGGAGCAGAGC GTTCTCTCC CAGCCTGGTGAT CCTCAGTTTCTAAGCATCCAAGAGGGAGAAAATCTCACTGTGTACTGCAACTCCTCAAGTGTTTTTTCCAGCTTAC ACATCACTG ACAGGCCTCTAC AATGGTACAGACAGGAGCCTGGGGAAGGTCCTGTCCTCCTGGTGACAGTAGTTACGGGTGGAGAAGTGAAGAAG CAGCC CTCTGTGCAGGA CTGAAGAGACTAACCTTTCAGTTTGGTGATGCAAGAAAGGACA G hTRAV28 hTRAV28 156 ATGAAGGCATTAATAGGAATCTTGCTGGGCTTCCTGTGGATACAGATTTGCTCGCAAATGAAAGTGGAGCAGAG GCCACCTAT GCCTGAGGACTC TCCTCAGGTCCTGATCCTCCAAGAGGGAAGAAATTCATTCCTGGTGTGCAGTTGTTCTATTTACATGATCCGTGTG ACATCAGAT AGCTATTTACTTC CAGTGGTTTCATCAAAAGCCTGGAGGACCCCTCATGTCCTTATTTAACATTAATTCAGGAATACAGCAAAAAAGA TCCCA TGTGCTGTGGGG AGACTAAAATCCGCAGTCAAAGCTGAGGAACTTTATG A hTRAV29 hTRAV29 157 ATGGCCATGCTCCTGGGGGCATCAGTGCTGATTCTGTGGCTTCAGCCAGACTGGGTAAACAGTCAACAGAAGAA TCTCTGCAC GCCTGGAGACTC TGATGACCAGCAAGTTAAGCAAAATTCACCATCCCTGAGCGTCCAGGAAGGAAGAATTTCTATTCTGAACTGTG ATTGTGCCC TGCAGTGTACTT ACTATACTAACAGCATGTTTGATTATTTCCTATGGTACAAAAAATACCCTGCTGAAGGTCCTACATTCCTGATATC TCCCA CTGTGCAGCAAG TATAAGTTCCATTAAGGATAAAAATGAAGATGGAAGATTCACTGTCTTCTTAAACAAAAGTGCCAAGCACCTC CG hTRAV30 hTRAV30 158 ATGGAGACTCTCCTGAAAGTGCTTTCAGGCACCTTGTTGTGGCAGTTGACCTGGGTGAGAAGCCAACAACCAGTG CCCTGTACC CAGTTACTCAGG CAGAGTCCTCAAGCCGTGATCCTCCGAGAAGGGGAAGATGCTGTCATCAACTGCAGTTCCTCCAAGGCTTTATAT TTACGGCCT AACCTACTTCTG TCTGTACACTGGTACAGGCAGAAGCATGGTGAAGCACCCGTCTTCCTGATGATATTACTGAAGGGTGGAGAACA CCCAGCT CGGCACAGAGA GAAGGGTCATGAAAAAATATCTGCTTCATTTAATGAAAAAAAGCAGCAAAGCT hTRAV31 hTRAV31 159 ATGACTGTTGGCAGCATATTACGGGCACTCATGGCCTCTGCCTTCCTTGCATGTCACAGAGGGTCATTCAATCCC CTTATCATA GAAGACCTGCAA AACCAGCAATATCTACGCAGGAGGGTGAGACCGTGAAACTGGACTGTGCATACAAAACTAATATTGTATATTAC TCATCATCA CATATTTCTGTTG ATATTGTATTGGTACAAAAGGTCTCCCAATGGGAAGATTATTTTCCTCATTTATCAGCAAACAGATGCAGAAACC CAGCCA TCTCAAAGAGCC AATGCGACACAGGGTCAATATTCTGTGAGCTTCCAGAAAACAACTAAAACTATTCAG hTRAV32 hTRAV32 160 ATGGCAAGAAGAATGGAAAAGTCCCTGGGAGCTTTATTCAAATTCAGCTGAAGCTGGCCAAGAAAAGGATGTGA TCCCTGCAT CCAGGAGACTCA TACAGAGTTATTCAAATCTAAATGTCTAGGAGAGAGAAATGGCCGTTATTAATGACAGTTATACAGATGGAGCTT ATTACAGCC TTCCTGTACTTCT TGAATTATTTCTGTTGGTACAAGAAGAAAACGGGGAAGGCCCTAATATCTTAATGGAGATTCATTCAAATGTGGA ACCCAA GTGCAGTGAGAA TAGAAAACAGGACAGAAGGCTCACTGTACTGTTGAATAAAAATGCTAAACATGTC CACA hTRAV33 hTRAV33 161 ATGCTCTGCCCTGGCCTGCTGTGGGCATTCGTGGTCCCCTTTGGCTTCAGATCCAGCATGGCTCAGAAAGTAACC ACCTCACCA TGACTCAGCCAA CAAGTTCAGACCACAGTAACTAGGCAGAAAGGAGTAGCTGTGACCTTGGACTGCATGTTTGAAACCAGATAGAA TCAATTCCT GTACTTCTGTGCT TTCGTACACTTTATACTGGTACAAGCAACAAGCAACCTCCCAGTGAAGAGATGGTTTTCCTTATTCATCAGGGTT TAAAAC CTCAGGAATCC ATTCTAAGTCAAATGCAAAGCCTGTGAACTTTGAAAAAAAGAAAAAGTTCATCA hTRAV34 hTRAV34 162 ATGGAGACTGTTCTGCAAGTACTCCTAGGGATATTGGGGTTCCAAGCAGCCTGGGTCAGTAGCCAAGAACTGGA TCCCTGCAT CCCAGCCATGCA GCAGAGTCCTCAGTCCTTGATCGTCCAAGAGGGAAAGAATCTCACCATAAACTGCACGTCATCAAAGACGTTAT ATCACAGCC GGCATCTACCTC ATGGCTTATACTGGTATAAGCAAAAGTATGGTGAAGGTCTTATCTTCTTGATGATGCTACAGAAAGGTGGGGAA TCCCAG TGTGGAGCAGAC GAGAAAAGTCATGAAAAGATAACTGCCAAGTTGGATGAGAAAAAGCAGCAAAGT A hTRAV35 hTRAV35 163 ATGCTCCTTGAACATTTATTAATAATCTTGTGGATGCAGCTGACATGGGTCAGTGGTCAACAGCTGAATCAGAGT CTTCCTGAA ACCTAGTGATGT CCTCAATCTATGTTTATCCAGGAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAACACCTGG TATCTCAGC AGGCATCTACTT CTATGGTACAAGCAGGAACCTGGGGAAGGTCCTGTCCTCTTGATAGCCTTATATAAGGCTGGTGAATTGACCTCA ATCCAT CTGTGCTGGGCA AATGGAAGACTGACTGCTCAGTTTGGTATAACCAGAAAGGACAG G hTRAV36 hTRAV36 164 ATGATGAAGTGTCCACAGGCTTTACTAGCTATCTTTTGGCTTCTACTGAGCTGGGTGAGCAGTGAAGACAAGGTG TCCTGAACA ACCGGAGACTCG GTACAAAGCCCTCTATCTCTGGTTGTCCACGAGGGAGACACCGTAACTCTCAATTGCAGTTATGAAGTGACTAAC TCACAGCCA GCCATCTACCTC TTTCGAAGCCTACTATGGTACAAGCAGGAAAAGAAAGCTCCCACATTTCTATTTATGCTAACTTCAAGTGGAATT CCCAG TGTGCTGTGGAG GAAAAGAAGTCAGGAAGACTAAGTAGCATATTAGATAAGAAAGAACTTTCCAGCA G hTRAV37 hTRAV37 165 ATGGAAACTCCACTGAGCACTCTGCTGCTGCTCCTCTGTGTGCAGCTGACCTGGTCAAATGGACAACTGCCAGTG TCCCTGCAC CTCCATGACTCA GAACAGAATGCTCCTTCCCTGAAAGTCAAGGAAGGTGACAGCGTCACACTGAACTGCAGTTACAGAGACAGCCC ATACAGGAT ACCACATTCTTCT TTCAGATTTCTTCAGTGGTTCAGGCAGGATCCTGAGGAAGGCCTCATTTCCCTGATACAAATGCTATCAACTGTG TCCCAG GCGCAGCAAGCA AGAGAGAAGATCAGTGGAAGATTCACAGCCAGGCTTAAAAAAGGAGACCAGCACATT hTRAV38-1 hTRAV38 166 ATGACACGAGTTAGCTTGCTGTGGGCAGTCGTGGTCTCCACCTGTCTTGAATCCGGCATGGCCCAGACAGTCACT CAAGATCTC GGGACACTGCGA CAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACTGTGACCCTGAGTTGCACATATGACACCAGTGAGAA AGACTCACA TGTATTTCTGTGC TAATTATTATTTGTTCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAA GCTGG TTTCATGAAGCA GCAACAGAATGCAACGGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCT hTRAV38-2 hTRAV38 167 ATGGCATGCCCTGGCTTCCTGTGGGCACTTGTGATCTCCACCTGTCTTGAATTTAGCATGGCTCAGACAGTCACTC CAAGATCTC GGGATGCCGCGA AGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACCGTGACCCTGAGCTGCACATATGACACCAGTGAGAGT AGACTCACA TGTATTTCTGTGC GATTATTATTTATTCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAG GCTGG TTATAGGAGCG CAACAGAATGCAACAGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCT hTRAV39 hTRAV39 168 ATGAAGAAGCTACTAGCAATGATTCTGTGGCTTCAACTAGACCGGTTAAGTGGAGAGCTGAAAGTGGAACAAAA CCGTCTCAG CAGCTGCCGTGC CCCTCTGTTCCTGAGCATGCAGGAGGGAAAAAACTATACCATCTACTGCAATTATTCAACCACTTCAGACAGACT CACCCTCCA ATGACCTCTCTG GTATTGGTACAGGCAGGATCCTGGGAAAAGTCTGGAATCTCTGTTTGTGTTGCTATCAAATGGAGCAGTGAAGCA CATCA CCACCTACTTCT GGAGGGACGATTAATGGCCTCACTTGATACCAAAGC GTGCCGTGGACA hTRAV40 hTRAV40 169 ATGAACTCCTCTCTGGACTTTCTAATTCTGATCTTAATGTTTGGAGGAACCAGCAGCAATTCAGTCAAGCAGACG CCATTGTGA TATCAGACTCAG GGCCAAATAACCGTCTCGGAGGGAGCATCTGTGACTATGAACTGCACATACACATCCACGGGGTACCCTACCCTT AATATTCAG CCGTGTACTACT TTCTGGTATGTGGAATACCCCAGCAAACCTCTGCAGCTTCTTCAGAGAGAGACAATGGAAAACAGCAAAAACTT TCCAGG GTCTTCTGGGAG CGGAGGCGGAAATATTAAAGACAAAAACTCCC A hTRAV41 hTRAV10 170 ATGGTGAAGATCCGGCAATTTTTGTTGGCTATTTTGTGGCTTCAGCTAAGCTGTGTAAGTGCCGCCAAAAATGAA CTGCACATC TCCCAGAGACTC GTGGAGCAGAGTCCTCAGAACCTGACTGCCCAGGAAGGAGAATTTATCACAATCAACTGCAGTTACTCGGTAGG ACAGCCTCC TGCCGTCTACAT AATAAGTGCCTTACACTGGCTGCAACAGCATCCAGGAGGAGGCATTGTTTCCTTGTTTATGCTGAGCTCAGGGAA CA CTGTGCTGTCAG GAAGAAGCATGGAAGATTAATTGCCACAATAAACATACAGGAAAAGCACAGCTCC A

TABLE 16 TCR β chain V segments and binding sites for primers presented in Table 4 and Table 10. The sequence for each V segments presented in this table consists of three parts (listed from 5′ to 3′): Sequence upstream of primer binding site, sequence of the primer binding site and sequence downstream of the primer binding site. V SEQ Primer hTRbV sequence segment Primer ID binding site downstream of name name NO hTRBV sequence upstream of primer binding site within hTRbV primer binding site hTRBV01 hTRBV01 171 ATGGGCTGAAGTCTCCACTGTGGTGTGGTCCATTGTCTCAGGCTCCATGGATACTGGAATTACCCAGACACCAAA GTGGTCGCA CTCAGCTGCGTA ATACCTGGTCACAGCAATGGGGAGTAAAAGGACAATGAAACGTGAGCATCTGGGACATGATTCTATGTATTGGT CTGCAGCAA TCTCTGCACCAG ACAGACAGAAAGCTAAGAAATCCCTGGAGTTCATGTTTTACTACAACTGTAAGGAATTCATTGAAAACAAGACT GAAGA CAGCCAAGA GTGCCAAATCACTTCACACCTGAATGCCCTGACAGCTCTCGCTTATACCTTCAT hTRBV02 hTRBV02 172 ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCAGGACTCACAGAACCTGAAGTCACCCAG GATCCGGTC CTCAGCCATGTA ACTCCCAGCCATCAGGTCACACAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATAC CACAAAGCT CTTCTGTGCCAG TTCTATTGGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTTCTGGTTTCCTTTTATAATAATGAAATCTCAGAG GGAGGA CAGTGAAGC AAGTCTGAAATATTCGATGATCAATTCTCAGTTGAAAGGCCTGATGGATCAAATTTCACTCTGAA hTRBV03-1 hTRBV03-1 173 ATGGGCTGCAGGCTCCTCTGCTGTGTGGTCTTCTGCCTCCTCCAAGCAGGTCCCTTGGACACAGCTGTTTCCCAGA CATCAATTC CTCTGCTGTGTAT CTCCAAAATACCTGGTCACACAGATGGGAAACGACAAGTCCATTAAATGTGAACAAAATCTGGGCCATGATACT CCTGGAGCT TTCTGTGCCAGC ATGTATTGGTATAAACAGGACTCTAAGAAATTTCTGAAGATAATGTTTAGCTACAATAATAAGGAGCTCATTATA TGGTGA AGCCAAGA AATGAAACAGTTCCAAATCGCTTCTCACCTAAATCTCCAGACAAAGCTCACTTAAATCTTCA hTRBV03-2 hTRBV03-1 174 ATGGGCTGCAGGCTCCTCTGCTATGTGGCCCTCTGCCTCCTGCAAGCAGGATCCACTGGACACAGCCGTTTCCCA CATCAATTC CTCTGCTGTGTAT GACTCCAAAATACCTGGTCACACAGATGGGAAAAAAGGAGTCTCTTAAATGAGAACAAAATCTGGGCCATAATG CCTGGAGCT TTCTGTGCCAGC CTATGTATTGGTATAAACAGGACTCTAAGAAATTTCTGAAGACAATGTTTATCTACAGTAACAAGGAGCCAATTT TGGTGA AGCCAAGA TAAATGAAACAGTTCCAAATCGCTTCTCACCTGACTCTCCAGACAAAGCTCATTTAAATCTTCA hTRBV04-1 hTRBV04-1 175 ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCAGTTCCCATAGACACTGAAGTTACCCAG TTCACCTAC AAGACTCAGCCC ACACCAAAACACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATATGGGGCACAGGGC ACGCCCTGC TGTATCTCTGCG TATGTATTGGTACAAGCAGAAAGCTAAGAAGCCACCGGAGCTCATGTTTGTCTACAGCTATGAGAAACTCTCTAT AGCCAG CCAGCAGCCAAG AAATGAAAGTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCTCTTAAACC A hTRBV04-2 hTRBV04-2 176 ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCGGTCCCCATGGAAACGGGAGTTACGCAG TTCACCTAC AAGACTCGGCCC ACACCAAGACACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATCTGGGGCATAACGC ACACCCTGC TGTATCTCTGTGC TATGTATTGGTACAAGCAAAGTGCTAAGAAGCCACTGGAGCTCATGTTTGTCTACAACTTTAAAGAACAGACTGA AGCCAG CAGCAGCCAAGA AAACAACAGTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCACTTATTCC hTRBV04-3 hTRBV04-2 177 ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCGGGTGAGTTGGTCCCCATGGAAACGGGA TTCACCTAC AAGACTCGGCCC GTTACGCAGACACCAAGACACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATCTGGG ACACCCTGC TGTATCTCTGCG TCATAACGCTATGTATTGGTACAAGCAAAGTGCTAAGAAGCCACTGGAGCTCATGTTTGTCTACAGTCTTGAAGA AGCCAG CCAGCAGCCAAG ACGGGTTGAAAACAACAGTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCACTTATTCC A hTRBV05-1 hTRBV05-1 178 ATGGGCTCCAGGCTGCTCTGTTGGGTGCTGCTTTGTCTCCTGGGAGCAGGCCCAGTAAAGGCTGGAGTCACTCAA GAATGTGAG GGGACTCGGCCC ACTCCAAGATATCTGATCAAAACGAGAGGACAGCAAGTGACACTGAGCTGCTCCCCTATCTCTGGGCATAGGAG CACCTTGGA TTTATCTTTGCGC TGTATCCTGGTACCAACAGACCCCAGGACAGGGCCTTCAGTTCCTCTTTGAATACTTCAGTGAGACACAGAGAAA GCTGG CAGCAGCTTGG CAAAGGAAACTTCCCTGGTCGATTCTCAGGGCGCCAGTTCTCTAACTCTCGCTCTGAGAT hTRBV05-2 hTRBV05-2 179 ATGGGCTCCGGACTCCTCTGCTGGACGCTGCTTTGTTTCCTGGGAGCAGGCCCAGTGGAGGCTGGAATCACCCAA TACTGAGTC GGACTCAGCCCT GCTCCAAGACACCTGATCAAAACAAGAGACCAGCAAGTGACACTGAGATGCTCCCCTGCCTCTGGGCATAACTG AAACACGGA GTATCTCTGTGC TGTGTCCTGGTACCTACGAACTCCAAGTCAGCCCCTCTAGTTATTGTTACAATATTGTAATAGGTTACAAAGAGC GCTAGG CAGCAACTTG AAAAGGAAACTTGCCTAATTGATTCTCAGCTCACCACGTCCATAACTAT hTRBV05-3 hTRBV05-3 180 ATGGGCCCCGGGCTCCTCTGCTGGGAACTGCTTTATCTCCTGGGAGCAGGCCCAGTGGAGGCTGGAGTCACCCAA GCTCTGAGA TGGAGCTGGGGG AGTCCCACACACCTGATCAAAACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTATCTCTGGGCACAGCAG TGAATGTGA ACTCGGCCCTGT TGTGTCCTGGTACCAACAGGCCCCGGGTCAGGGGCCCCAGTTTATCTTTGAATATGCTAATGAGTTAAGGAGATC GTGCCT ATCTCTGTGCCA AGAAGGAAACTTCCCTAATCGATTCTCAGGGCGCCAGTTCCATGACTGTT GAAGCTTGG hTRBV05-4 hTRBV05-4 181 ATGGGCCCTGGGCTCCTCTGCTGGGTGCTGCTTTGTCTCCTGGGAGCAGGCTCAGTGGAGACTGGAGTCACCCAA CTGAGCTGA GGAGCTGGACGA AGTCCCACACACCTGATCAAAACGAGAGGACAGCAAGTGACTCTGAGATGCTCTTCTCAGTCTGGGCACAACAC ATGTGAACG CTCGGCCCTGTA TGTGTCCTGGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTTATCTTTCAGTATTATAGGGAGGAAGAGAATGG CCTT TCTCTGTGCCAG CAGAGGAAACTTCCCTCCTAGATTCTCAGGTCTCCAGTTCCCTAATTATAGCT CAGCTTGG hTRBV05-5 hTRBV05-4 182 ATGGGCCCTGGGCTCCTCTGCTGGGTGCTGCTTTGTCTCCTGGGAGCAGGCCCAGTGGACGCTGGAGTCACCCAA CTGAGCTGA GTTGCTGGGGGA AGTCCCACACACCTGATCAAAACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTATCTCTGGGCACAAGAG ATGTGAACG CTCGGCCCTGTA TGTGTCCTGGTACCAACAGGTCCTGGGTCAGGGGCCCCAGTTTATCTTTCAGTATTATGAGAAAGAAGAGAGAG CCTT TCTCTGTGCCAG GAAGAGGAAACTTCCCTGATCGATTCTCAGCTCGCCAGTTCCCTAACTATAGCT CAGCTTGG hTRBV05-6 hTRBV05-4 183 ATGGGCCCCGGGCTCCTCTGCTGGGCACTGCTTTGTCTCCTGGGAGCAGGCTTAGTGGACGCTGGAGTCACCCAA CTGAGCTGA GTTGCTGGGGGA AGTCCCACACACCTGATCAAAACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTAAGTCTGGGCATGACAC ATGTGAACG CTCGGCCCTCTA TGTGTCCTGGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTTATCTTTCAGTATTATGAGGAGGAAGAGAGAC CCTT TCTCTGTGCCAG AGAGAGGCAACTTCCCTGATCGATTCTCAGGTCACCAGTTCCCTAACTATAGCT CAGCTTGG hTRBV05-7 hTRBV05-4 184 ATGGGCCCCGGGCTCCTCTGCTGGGTGCTGCTTTGTCCCCTAGGAGAAGGCCCAGTGGACGCTGGAGTCACCCAA CTGAGCTGA GTTGCTAGGGGA AGTCCCACACACCTGATCAAAACGAGAGGACAGCACGTGACTCTGAGATGCTCTCCTATCTCTGGGCACACCAG ATGTGAACG CTCGGCCCTCTA TGTGTCCTCGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTTATCTTTCAGTATTATGAGAAAGAAGAGAGAG CCTT TCTCTGTGCCAG GAAGAGGAAACTTCCCTGATCAATTCTCAGGTCACCAGTTCCCTAACTATAGCT CAGCTTGG hTRBV05-8 hTRBV05-4 185 ATGGGACCCAGGCTCCTCTTCTGGGCACTGCTTTGTCTCCTCGGAACAGGCCCAGTGGAGGCTGGAGTCACACAA CTGAGCTGA GGAGCTGGAGGA AGTCCCACACACCTGATCAAAACGAGAGGACAGCAAGCGACTCTGAGATGCTCTCCTATCTCTGGGCACACCAG ATGTGAACG CTCGGCCCTGTA TGTGTACTGGTACCAACAGGCCCTGGGTCTGGGCCTCCAGTTCCTCCTTTGGTATGACGAGGGTGAAGAGAGAAA CCTT TCTCTGTGCCAG CAGAGGAAACTTCCCTCCTAGATTTTCAGGTCGCCAGTTCCCTAATTATAGCT CAGCTTGG hTRBV06-1 hTRBV06-1 186 ATGAGCATCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAAGTCCAGTGAATGCTGGTGTCACTCAG GAGTTCTCG CGGCTGCTCCCT ACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATAACTC CTCAGGCTG CCCAGACATCTG CATGTACTGGTATCGACAAGACCCAGGCATGGGACTGAGGCTGATTTATTACTCAGCTTCTGAGGGTACCACTGA GAGT TGTACTTCTGTGC CAAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATTAAACAAACGG CAGCAGTGAAGC hTRBV06-2 hTRBV06-2 187 ATGAGCCTCGGGCTCCTGTGCTGTGGGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAG CTGGGGTTG CCTCCCAAACAT ACCCCAAAATTCCGGGTCCTGAAGACAGGACAGAGCATGACACTGCTGTGTGCCCAGGATATGAACCATGAATA GAGTCGGCT CTGTGTACTTCTG CATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGAGGGTACAACTG GCTC TGCCAGCAGTTA CCAAAGGAGAGGTCCCTGATGGCTACAATGTCTCCAGATTAAAAAAACAGAATTTCCTG CTC hTRBV06-3 hTRBV06-2 188 ATGAGCCTCGGGCTCCTGTGCTGTGGGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAG CTGGGGTTG CCTCCCAAACAT ACCCCAAAATTCCGGGTCCTGAAGACAGGACAGAGCATGACACTGCTGTGTGCCCAGGATATGAACCATGAATA GAGTCGGCT CTGTGTACTTCTG CATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGAGGGTACAACTG GCTC TGCCAGCAGTTA CCAAAGGAGAGGTCCCTGATGGCTACAATGTCTCCAGATTAAAAAAACAGAATTTCCTG CTC hTRBV06-4 hTRBV06-4 189 ATGAGAATCAGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGATTGCTGGGATCACCCAG CCCCTCACG TACCCTCTCAGA GCACCAACATCTCAGATCCTGGCAGCAGGACGGCGCATGACACTGAGATGTACCCAGGATATGAGACATAATGC TTGGCGTCT CATCTGTGTACTT CATGTACTGGTATAGACAAGATCTAGGACTGGGGCTAAGGCTCATCCATTATTCAAATACTGCAGGTACCACTGG GCTG CTGTGCCAGCAG CAAAGGAGAAGTCCCTGATGGTTATAGTGTCTCCAGAGCAAACACAGATGATTTC TGACTC hTRBV06-5 hTRBV06-5 190 ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAG TCCCGCTCA TGCTCCCTCCCA ACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATA GGCTGCTGT GACATCTGTGTA CATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGA CGGC CTTCTGTGCCAG CCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATT CAGTTACTC hTRBV06-6 hTRBV06-6 191 ATGAGCATCAGCCTCCTGTGCTGTGCAGCCTTTCCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAG GATTTCCCG TGGCTGCTCCCT ACCCCAAAATTCCGCATCCTGAAGATAGGACAGAGCATGACACTGCAGTGTACCCAGGATATGAACCATAACTA CTCAGGCTG CCCAGACATCTG CATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAAGCTGATTTATTATTCAGTTGGTGCTGGTATCACTGA GAGT TGTACTTCTGTGC TAAAGGAGAAGTCCCGAATGGCTACAACGTCTCCAGATCAACCACAGAG CAGCAGTTACTC hTRBV06-7 hTRBV06-7 192 ATGAGCCTCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAGGTCCAATGAATGCTGGTGTCACTCAGA TCCCCCTCA GCTCCCTCTCAG CCCCAAAATTCCACGTCCTGAAGACAGGACAGAGCATGACTCTGCTGTGTGCCCAGGATATGAACCATGAATAC AGCTGGAGT ACTTCTGTTTACT ATGTATCGGTATCGACAAGACCCAGGCAAGGGGCTGAGGCTGATTTACTACTCAGTTGCTGCTGCTCTCACTGAC CAGCT TCTGTGCCAGCA AAAGGAGAAGTTCCCAATGGCTACAATGTCTCCAGATCAAACACAGAGGATT GTTACTC hTRBV06-8 hTRBV06-8 193 ATGAGCCTCGGGCTCCTGTGCTGTGCGGCCTTTTCTCTCCTGTGGGCAGGTCCCGTGAATGCTGGTGTCACTCAGA TCCCACTCA TGCTCCCTCCCA CCCCAAAATTCCACATCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGGATAC GGCTGGTGT GACATCTGTGTA ATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGACTGATTTACTACTCAGCTGCTGCTGGTACTACTGAC CGGC CTTGTGTGCCAG AAAGAAGTCCCCAATGGCTACAATGTCTCTAGATTAAACACAGAGGATT CAGTTACTC hTRBV06-9 hTRBV06-6 194 ATGAGCATCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGAGGGTCCAGTGAATGCTGGTGTCACTCAG GATTTCCCG CAGCTGCTCCCT ACCCCAAAATTCCACATCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGGATA CTCAGGCTG CCCAGACATCTG CTTGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCGCATTCATTACTCAGTTGCTGCTGGTATCACTGA GAGT TATACTTCTGTGC CAAAGGAGAAGTCCCCGATGGCTACAATGTATCCAGATCAAACACAGAG CAGCAGTTATTC hTRBV07-1 hTRBV07-1 195 ATGGGCACAAGGCTCCTCTGCTGGGCAGCCATATGTCTCCTGGGGGCAGATCACACAGGTGCTGGAGTCTCCCA CTCTGAAGT GCAGGGGGACTT GTCCCTGAGACACAAGGTAGCAAAGAAGGGAAAGGATGTAGCTCTCAGATATGATCCAATTTCAGGTCATAATG TCCAGCGCA GGCTGTGTATCT CCCTTTATTGGTACCGACAGAGCCTGGGGCAGGGCCTGGAGTTTCCAATTTACTTCCAAGGCAAGGATGCAGCAG CACA CTGTGCCAGCAG ACAAATCGGGGCTTCCCCGTGATCGGTTCTCTGCACAGAGGTCTGAGGGATCCATCTCCA CTCAGC hTRBV07-2 hTRBV07-2 196 ATGGGCACCAGGCTCCTCTTCTGGGTGGCCTTCTGTCTCCTGGGGGCAGATCACACAGGAGCTGGAGTCTCCCAG GATCCAGCG GACTCGGCCGTG TCCCCCAGTAACAAGGTCACAGAGAAGGGAAAGGATGTAGAGCTCAGGTGTGATCCAATTTCAGGTCATACTGC CACACAGCA TATCTCTGTGCC CCTTTACTGGTACCGACAGAGCCTGGGGCAGGGCCTGGAGTTTTTAATTTACTTCCAAGGCAACAGTGCACCAGA GGAG AGCAGCTTAGC CAAATCAGGGCTGCCCAGTGATCGCTTCTCTGCAGAGAGGACTGGGGGATCCGTCTCCACTCTGAC hTRBV07-3 hTRBV07-5 197 ATGGGCACCAGGCTCCTCTGCTGGGCAGCCCTGTGCCTCCTGGGGGCAGATCACACAGGTGCTGGAGTCTCCCAG ACTCTGAAG GCGGGGGGACTC ACCCCCAGTAACAAGGTCACAGAGAAGGGAAAATATGTAGAGCTCAGGTGTGATCCAATTTCAGGTCATACTGC ATCCAGCGC AGCCGTGTATCT CCTTTACTGGTACCGACAAAGCCTGGGGCAGGGCCCAGAGTTTCTAATTTACTTCCAAGGCACGGGTGCGGCAG ACAGA CTGTGCCAGCAG ATGACTCAGGGCTGCCCAACGATCGGTTCTTTGCAGTCAGGCCTGAGGGATCCGTCTCT CTTAAC hTRBV07-4 hTRBV07-5 198 ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATCACACAGGTGCTGGAGTCTCCCAG ACTCTGAAG GCAGGGGGACTC TCCCCAAGGTACAAAGTCGCAAAGAGGGGACGGGATGTAGCTCTCAGGTGTGATTCAATTTCGGGTCATGTAAC ATCCAGCGC AGCTGTGTATCT CCTTTATTGGTACCGACAGACCCTGGGGCAGGGCTCAGAGGTTCTGACTTACTCCCAGAGTGATGCTCAACGAGA ACAGA CTGTGCCAGCAG CAAATCAGGGCGGCCCAGTGGTCGGTTCTCTGCAGAGAGGCCTGAGAGATCCGTCTCC CTTAGC hTRBV07-5 hTRBV07-5 199 ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATCACACAGGTGCTGGAGTCTCCCAG AGATCCAGC GCGACTCGGCTG TCCCCAAGGTACGAAGTCACACAGAGGGGACAGGATGTAGCTCCCAGGTGTGATCCAATTTCGGGTCAGGTAAC GCACAGAGC TGTATCTCTGTGC CCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCAAGAGTTTCTGACTTCCTTCCAGGATGAAACTCAACAAGA AAGG CAGAAGCTTAG TAAATCAGGGCTGCTCAGTGATCAATTCTCCACAGAGAGGTCTGAGGATCTTTCTCCACCTGA hTRBV07-6 hTRBV07-6 200 ATGGGCACCAGTCTCCTATGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATCACACAGGTGCTGGAGTCTCCCAG CAGCGCACA CGGCCATGTATC TCTCCCAGGTACAAAGTCACAAAGAGGGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCATGTATCC GAGCAGCGG GCTGTGCCAGCA CTTTATTGGTACCGACAGGCCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCAATTATGAAGCCCAACAAGAC GACT GCTTAGC AAATCAGGGCTGCCCAATGATCGGTTCTCTGCAGAGAGGCCTGAGGGATCCATCTCCACTCTGACGATC hTRBV07-7 hTRBV07-6 201 ATGGGTACCAGTCTCCTATGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATCACACAGGTGCTGGAGTCTCCCAG CAGCGCACA CAGCCATGTATC TCTCCCAGGTACAAAGTCACAAAGAGGGGACAGGATGTAACTCTCAGGTGTGATCCAATTTCGAGTCATGCAAC GAGCAGCGG GCTGTGCCAGCA CCTTTATTGGTATCAACAGGCCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCAATTATGAAGCTCAACCAGA GACT GCTTAGC CAAATCAGGGCTGCCCAGTGATCGGTTCTCTGCAGAGAGGCCTGAGGGATCCATCTCCACTCTGACGATT hTRBV07-8 hTRBV07-2 202 ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATCACACAGGTGCTGGAGTCTCCCAG GATCCAGCG GACTCCGCCGTG TCCCCTAGGTACAAAGTCGCAAAGAGAGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCATGTATCC CACACAGCA TATCTCTGTGCC CTTTTTTGGTACCAACAGGCCCTGGGGCAGGGGCCAGAGTTTCTGACTTATTTCCAGAATGAAGCTCAACTAGAC GGAG AGCAGCTTAGC AAATCGGGGCTGCCCAGTGATCGCTTCTTTGCAGAAAGGCCTGAGGGATCCGTCTCCACTCTGAA hTRBV07-9 hTRBV07-9  203 ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAG GAGATCCAG GGGACTCGGCCA AACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCG CGCACAGAG TGTATCTCTGTGC CCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGA CAGG CAGCAGCTTAGC AAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTG hTRBV08-1 hTRBV08-1 204 GAGGCAGGGATCAGCCAGATACCAAGATATCACAGACACACAGGGAAAAAGATCATCCTGAAATATGCTCAGA CCCTCAACC GCACCAGCCAGA TTAGGAACCATTATTCAGTGTTCTGTTATCAATAAGACCAAGAATAGGGGCTGAGGCTGATCCATTATTCAGGTA CTGGAGTCT CCTCTGTACCTCT GTATTGGCAGCATGACCAAAGGCGGTGCCAAGGAAGGGTACAATGTCTCTGGAAACAAGCTCAAGCATTTT ACTA GTGGCAGTGCAT C hTRBV08-2 hTRBV08-2 205 ATGAACCCCAAACTCTTCTGTGTGACCCTTTGTCTCCTGGGAGCAGGCTCTATTGATGCTGGGATCACCCAGATG TCCCCAATC GCACCAGCCAGA CCAAGATATCACATTGTACAGAAGAAAGAGATGATCCTGGAATGTGCTCAGGTTAGGAACAGTGTTCTGATATC CTGGCATCC CCTATCTGTACC GACAGGACCCAAGACGGGGGCTGAAGCTTATCCACTATTCAGGCAGTGGTCACAGCAGGACCAAAGTTGATGTC ACCA ACTGTGGCAGCA ACAGAGGGGTACTGTGTTTCTTGAAACAAGCTTGAGCATT CATC hTRBV09 hTRBV09 206 ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAA CTAAACCTG GGGGGACTCAGC ACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTC AGCTCTCTG TTTGTATTTCTGT TGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGAGAG GAGCT GCCAGCAGCGTA CAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAA G hTRBV10-1 hTRBV10-1 207 ATGGGCACGAGGCTCTTCTTCTATGTGGCCCTTTGTCTGCTGTGGGCAGGACACAGGGATGCTGAAATCACCCAG CCCTCACTC CTCCTCCCAGAC AGCCCAAGACACAAGATCACAGAGACAGGAAGGCAGGTGACCTTGGCGTGTCACCAGACTTGGAACCACAACA TGGAGTCTG ATCTGTATATTTC ATATGTTCTGGTATCGACAAGACCTGGGACATGGGCTGAGGCTGATCCATTACTCATATGGTGTTCAAGACACTA CTGC TGCGCCAGCAGT ACAAAGGAGAAGTCTCAGATGGCTACAGTGTCTCTAGATCAAACACAGAGGACCTCC GAGTC hTRBV10-2 hTRBV10-2 208 ATGGGCACCAGGCTCTTCTTCTATGTGGCCCTTTGTCTGCTGTGGGCAGGACACAGGGATGCTGGAATCACCCAG CCCTCACTC CCGCTCCCAGAC AGCCCAAGATACAAGATCACAGAGACAGGAAGGCAGGTGACCTTGATGTGTCACCAGACTTGGAGCCACAGCTA TGGAGTCAG ATCTGTGTATTTC TATGTTCTGGTATCGACAAGACCTGGGACATGGGCTGAGGCTGATCTATTACTCAGCAGCTGCTGATATTACAGA CTAC TGCGCCAGCAGT TAAAGGAGAAGTCCCCGATGGCTATGTTGTCTCCAGATCCAAGACAGAGAATTTCC GAGTC hTRBV10-3 hTRBV10-3 209 ATGGGCACAAGGTTGTTCTTCTATGTGGCCCTTTGTCTCCTGTGGACAGGACACATGGATGCTGGAATCACCCAG TCCTCACTC CAGCTCCCAGAC AGCCCAAGACACAAGGTCACAGAGACAGGAACACCAGTGACTCTGAGATGTCACCAGACTGAGAACCACCGCT TGGAGTCCG ATCTGTGTACTTC ATATGTACTGGTATCGACAAGACCCGGGGCATGGGCTGAGGCTGATCCATTACTCATATGGTGTTAAAGATACTG CTAC TGTGCCATCAGT ACAAAGGAGAAGTCTCAGATGGCTATAGTGTCTCTAGATCAAAGACAGAGGATTTCC GAGTC hTRBV11-1 hTRBV11-1 210 ATGAGCACCAGGCTTCTCTGCTGGATGGCCCTCTGTCTCCTGGGGGCAGAACTCTCAGAAGCTGAAGTTGCCCAG CCACTCTCA GAGCTTGGGGAC TCCCCCAGATATAAGATTACAGAGAAAAGCCAGGCTGTGGCTTTTTGGTGTGATCCTATTTCTGGCCATGCTACC AGATCCAGC TCGGCCATGTAT CTTTACTGGTACCGGCAGATCCTGGGACAGGGCCCGGAGCTTCTGGTTCAATTTCAGGATGAGAGTGTAGTAGAT CTGCA CTCTGTGCCAGC GATTCACAGTTGCCTAAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACT AGCTTAGC hTRBV11-2 hTRBV11-1 211 ATGGGCACCAGGCTCCTCTGCTGGGCGGCCCTCTGTCTCCTGGGAGCAGAACTCACAGAAGCTGGAGTTGCCCA CCACTCTCA AAGCTTGAGGAC GTCTCCCAGATATAAGATTATAGAGAAAAGGCAGAGTGTGGCTTTTTGGTGCAATCCTATATCTGGCCATGCTAC AGATCCAGC TCGGCCGTGTAT CCTTTACTGGTACCAGCAGATCCTGGGACAGGGCCCAAAGCTTCTGATTCAGTTTCAGAATAACGGTGTAGTGGA CTGCA CTCTGTGCCAGC TGATTCACAGTTGCCTAAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACT AGCTTAGA hTRBV11-3 hTRBV11-1 212 ATGGGTACCAGGCTCCTCTGCTGGGTGGCCTTCTGTCTCCTGGTGGAAGAACTCATAGAAGCTGGAGTGGTTCAG CCACTCTCA GAGCTTGGGGAC TCTCCCAGATATAAGATTATAGAGAAAAAACAGCCTGTGGCTTTTTGGTGCAATCCTATTTCTGGCCACAATACC AGATCCAGC TCGGCCGTGTAT CTTTACTGGTACCTGCAGAACTTGGGACAGGGCCCGGAGCTTCTGATTCGATATGAGAATGAGGAAGCAGTAGA CTGCA CTCTGTGCCAGC CGATTCACAGTTGCCTAAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACT AGCTTAGA hTRBV12-1 hTRBV12-1 213 ATGGGCTCTTGGACCCTCTGTGTGTCCCTTTATATCCTGGTAGCGACACACACAGATGCTGGTGTTATCCAGTCAC GAGGATCCA GGGACTTGGGCC CCAGGCACAAAGTGACAGAGATGGGACAATCAGTAACTCTGAGATGCGAACCAATTTCAGGCCACAATGATCTT GCCCATGGA TATATTTCTGTGC CTCTGGTACAGACAGACCTTTGTGCAGGGACTGGAATTGCTGAATTACTTCTGCAGCTGGACCCTCGTAGATGAC ACCCA CAGCAGCTTTGC TCAGGAGTGTCCAAGGATTGATTCTCAGCACAGATGCCTGATGTATCATTCTCCACTCT hTRBV12-2 hTRBV12-2 214 ATGGACTCCTGGACCCTCTGTGTGTCCCTTTGTATCCTGGTAGCGACATGCACAGATGCTGGCATTATCCAGTCAC CTGAAGATC AGGGGGACTCGG CCAAGCATGAGGTGACAGAAATGGGACAAACAGTGACTCTGAGATGTGAGCCAATTTTTGGCCACAATTTCCTTT CAGCCTGCA CCGTGTATGTCT TCTGGTACAGAGATACCTTCGTGCAGGGACTGGAATTGCTGAGTTACTTCCGGAGCTGATCTATTATAGATAATG GAGC GTGCAAGTCGCT CAGGTATGCCCACAGAGCGATTCTCAGCTGAGAGGCCTGATGGATCATTCTCTACT TAGC hTRBV12-3 hTRBV12-3 215 ATGGACTCCTGGACCTTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCGAAGCATACAGATGCTGGAGTTATCCAG CAGCCCTCA CAGCTGTGTACT TCACCCCGCCATGAGGTGACAGAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGCCACAACTC GAACCCAGG TCTGTGCCAGCA CCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTGCTCATTTACTTTAACAACAACGTTCCGATAGA GACT GTTTAGC TGATTCAGGGATGCCCGAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTGAAGATC hTRBV12-4 hTRBV12-3 216 ATGGGCTCCTGGACCCTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCAAAGCACACAGATGCTGGAGTTATCCAG CAGCCCTCA CAGCTGTGTACT TCACCCCGGCACGAGGTGACAGAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGACACGACTA GAACCCAGG TCTGTGCCAGCA CCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTGCTCATTTACTTTAACAACAACGTTCCGATAGA GACT GTTTAGC TGATTCAGGGATGCCCGAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTGAAGATC hTRBV12-5 hTRBV12-3 217 ATGGCCACCAGGCTCCTCTGCTGTGTGGTTCTTTGTCTCCTGGGAGAAGAGCTTATAGATGCTAGAGTCACCCAG CAGCCCTCA CAGCTGTGTATT ACACCAAGGCACAAGGTGACAGAGATGGGACAAGAAGTAACAATGAGATGTCAGCCAATTTTAGGCCACAATA GAACCCAGG TTTGTGCTAGTG CTGTTTTCTGGTACAGACAGACCATGATGCAAGGACTGGAGTTGCTGGCTTACTTCCGCAACCGGGCTCCTCTAG GACT GTTTGGT ATGATTCGGGGATGCCGAAGGATCGATTCTCAGCAGAGATGCCTGATGCAACTTTAGCCACTCTGAAGATC hTRBV13 hTRBV13 218 ATGCTTAGTCCTGACCTGCCTGACTCTGCCTGGAACACCAGGCTCCTCTGCCATGTCATGCTTTGTCTCCTGGGAG GAGCTCCTT CAGCCCTGTACT CAGTTTCAGTGGCTGCTGGAGTCATCCAGTCCCCAAGACATCTGATCAAAGAAAAGAGGGAAACAGCCACTCTG GGAGCTGGG TCTGTGCCAGCA AAATGCTATCCTATCCCTAGACACGACACTGTCTACTGGTACCAGCAGGGTCCAGGTCAGGACCCCCAGTTCCTC GGACT GCTTAGG ATTTCGTTTTATGAAAAGATGCAGAGCGATAAAGGAAGCATCCCTGATCGATTCTCAGCTCAACAGTTCAGTGAC TATCATTCTGAACTGAACAT hTRBV14 hTRBV14 219 ATGGTTTCCAGGCTTCTCAGTTTAGTGTCCCTTTGTCTCCTGGGAGCAAAGCACATAGAAGCTGGAGTTACTCAG GGTGCAGCC GATTCTGGAGTT TTCCCCAGCCACAGCGTAATAGAGAAGGGCCAGACTGTGACTCTGAGATGTGACCCAATTTCTGGACATGATAA TGCAGAACT TATTTCTGTGCCA TCTTTATTGGTATCGACGTGTTATGGGAAAAGAAATAAAATTTCTGTTACATTTTGTGAAAGAGTCTAAACAGGA GGAG GCAGCCAAGA TGAGTCCGGTATGCCCAACAATCGATTCTTAGCTGAAAGGACTGGAGGGACGTATTCTACTCTGAA hTRBV15 hTRBV15 220 ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACAGGTCATGGGGATGCCATGGTCATCCAG GACATCCGC GGGACACAGCCA AACCCAAGATACCAGGTTACCCAGTTTGGAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTC TCACCAGGC TGTACCTGTGTG ATGTACTGGTACCAGCAGAAGTCAAGTCAGGCCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTAACAAT CTGG CCACCAGCAGAG GAAGCAGACACCCCTGATAACTTCCAATCCAGGAGGCCGAACACTTCTTTCTGCTTTCTT A hTRBV16 hTRBV16 221 ATGAGCCCAATATTCACCTGCATCACAATCCTTTGTCTGCTGGCTGCAGGTTCTCCTGGTGAAGAAGTCGCCCAG TGAGATCCA GAGGATTCAGCA ACTCCAAAACATCTTGTCAGAGGGGAAGGACAGAAAGCAAAATTATATTGTGCCCCAATAAAAGGACACAGTTA GGCTACGAA GTGTATTTTTGTG TGTTTTTTGGTACCAACAGGTCCTGAAAAACGAGTTCAAGTTCTTGATTTCCTTCCAGAATGAAAATGTCTTTGAT GCTT CCAGCAGCCAAT GAAACAGGTATGCCCAAGGAAAGATTTTCAGCTAAGTGCCTCCCAAATTCACCCTGTAGCCT C hTRBV17 hTRBV17 222 ATGGATATCTGGCTCCTCTGCTGGGTGACCCTGTGTCTCTTGGCGGCAGGACACTCGGAGCCTGGAGTCAGCCAG GAAGATCCA AGGGACTCAGCC ACCCCCAGACACAAGGTCACCAACATGGGACAGGAGGTGATTCTGAGGTGCGATCCATCTTCTGGTCACATGTTT TCCCGCAGA GTGTATCTCTAC GTTCACTGGTACCGACAGAATCTGAGGCAAGAAATGAAGTTGCTGATTTCCTTCCAGTACCAAAACATTGCAGTT GCCG AGTAGCGGTGG GATTCAGGGATGCCCAAGGAACGATTCACAGCTGAAAGACCTAACGGAACGTCTTCCACGCT hTRBV18 hTRBV18 223 ATGGACACCAGAGTACTCTGCTGTGCGGTCATCTGTCTTCTGGGGGCAGGTCTCTCAAATGCCGGCGTCATGCAG GGATCCAGC AGATTCGGCAGC AACCCAAGACACCTGGTCAGGAGGAGGGGACAGGAGGCAAGACTGAGATGCAGCCCAATGAAAGGACACAGTC AGGTAGTGC TTATTTCTGTGCC ATGTTTACTGGTATCGGCAGCTCCCAGAGGAAGGTCTGAAATTCATGGTTTATCTCCAGAAAGAAAATATCATAG GAGG AGCTCACCACC ATGAGTCAGGAATGCCAAAGGAACGATTTTCTGCTGAATTTCCCAAAGAGGGCCCCAGCATCCTGA hTRBV19 hTRBV19 224 ATGAGCAACCAGGTGCTCTGCTGTGTGGTCCTTTGTTTCCTGGGAGCAAACACCGTGGATGGTGGAATCACTCAG CACTGTGAC AACCCGACAGCT TCCCCAAAGTACCTGTTCAGAAAGGAAGGACAGAATGTGACCCTGAGTTGTGAACAGAATTTGAACCACGATGC ATCGGCCCA TTCTATCTCTGTG CATGTACTGGTACCGACAGGACCCAGGGCAAGGGCTGAGATTGATCTACTACTCACAGATAGTAAATGACTTTC AAAG CCAGTAGTATAG AGAAAGGAGATATAGCTGAAGGGTACAGCGTCTCTCGGGAGAAGAAGGAATCCTTTCCTCT A hTRBV20 hTRBV20 225 ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGGTATAAGCCTCCTTCTACCTGGGAGCTTGGCAGGCTCCGGGCTTG CTGACAGTG CTGAAGACAGCA GTGCTGTCGTCTCTCAACATCCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTCCC ACCAGTGCC GCTTCTACATCT TGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTCATGCTGATGGCAACTTCCA CATC GCAGTGCTAGAG ATGAGGGCTCCAAGGCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACC A TTGTCCACT hTRBV21 hTRBV21 226 ATGTGCCTCAGACTTCTCTGCTGTGTGGCCATTTCTTTCTGGGGAGCCAGGCTCCACGGACACCAAGGTCACCCA GAGATCCAG GGGACACAGCAC GAGACCTAGACTTCTGGTCAAAGCAAGTGAACAGAAAGCAAAGATGGATTGTGTTCCTATAAAAGCACATAGTT TCCACGGAG TGTATTTCTGTGC ATGTTTACTGGTATCGTAAGAAGCTGGAAGAAGAGCTCAAGTTTTTGGTTTACTTTCAGAATGAAGAACTTATTC TCAG CAGCAGCAAAGC AGAAAGCAGAAATAATCAATGAGCGATTTTTAGCCCAATGCTCCAAAAACTCATCCTGTACCTTG hTRBV22 hTRBV22 227 ATGGGGAGCTGGGTCCTCTGCTATGTGACCCTGTGTCTCCTGGGAGCAGGACCCTTGGATGCTGACATCTATCAG GTGAAGTTG AACAGCTTTGTA ATGCCATTCCAGCTCACTGGGGCTGGATGGGATGTGACTCTGGAGTGGAAACGGAATTTGAGACACAATGACAT GCCCACACC CTTCTGTCCTGG GTACTGCTACTGGTACTGGCAGGACCCAAAGCAAAATCTGAGACTGATCTATTACTCAAGGGTTGAAAAGGATA AGCCA GAGCGCAC TTCAGAGAGGAGATCTAACTGAAGGCTACGTGTCTGCCAAGAGGAGAAGGGGCTATTTCTTCTCAGG hTRBV23 hTRBV23 228 ATGGGCACCAGGCTCCTCGGCTGTGCAGCCCTGTGTCTCCTGGCAGCAGACTCTTTTCATGCCAAAGTCACACAG CCTGGCAAT CCGGGAGACACG ACTCCAGGACATTTGGTCAAAGGAAAAGGACAGAAAACAAAGATGGATTGTACCCCCGAAAAAGGACATACTTT CCTGTCCTC GCACTGTATCTC TGTTTATTGGTATCAACAGAATCAGAATAAAGAGTTTATGCTTTTGATTTCCTTTCAGAATGAACAAGTTCTTCAA AGAA TGCGCCAGCAGT GAAACGGAGATGCACAAGAAGCGATTCTCATCTCAATGCCCCAAGAACGCACCCTGCAG CAATC hTRBV24 hTRBV24 229 ATGGCCTCCCTGCTCTTCTTCTGTGGGGCCTTTTATCTCCTGGGAACAGGGTCCATGGATGCTGATGTTACCCAGA GAGTCTGCC CAGCTCTTTACTT CCCCAAGGAATAGGATCACAAAGACAGGAAAGAGGATTATGCTGGAATGTTCTCAGACTAAGGGTCATGATAGA ATCCCCAAC CTGTGCCACCAG ATGTACTGGTATCGACAAGACCCAGGACTGGGCCTACGGTTGATCTATTACTCCTTTGATGTCAAAGATATAAAC CAGA TGATTTG AAAGGAGAGATCTCTGATGGATACAGTGTCTCTCGACAGGCACAGGCTAAATTCTCCCTGTCCCTA hTRBV25 hTRBV25 230 ATGACTATCAGGCTCCTCTGCTACATGGGCTTTTATTTTCTGGGGGCAGGCCTCATGGAAGCTGACATCTACCAG GGAGTCTGC TACCTCTCAGTA ACCCCAAGATACCTTGTTATAGGGACAGGAAAGAAGATCACTCTGGAATGTTCTCAAACCATGGGCCATGACAA CAGGCCCTC CCTCTGTGCCAG AATGTACTGGTATCAACAAGATCCAGGAATGGAACTACACCTCATCCACTATTCCTATGGAGTTAATTCCACAGA ACA CAGTGAATA GAAGGGAGATCTTTCCTCTGAGTCAACAGTCTCCAGAATAAGGACGGAGCATTTTCCCCTGACCCT hTRBV26 hTRBV26 231 ATGAGCAACAGGCTTCTCTGCTGTGTGATCATTTGTCTCCTAAGAGCAGGCCTCAAGGATGCTGTAGTTACACAA GAAGTCTGC ACATCTGTGTAT TTCCCAAGACACAGAATCATTGGGACAGGAAAGGAATTCATTCTACAGTGTTCCCAGAATATGAATCATGTTACA CAGCACCAA CTCTATGCCAGC ATGTACTGGTATCGACAGGACCCAGGACTTGGACTGAAGCTGGTCTATTATTCACCTGGCACTGGGAGCACTGAA CCAG AGTTCATC AAAGGAGATATCTCTGAGGGGTATCATGTTTCTTGAAATACTATAGCATCTTTTCCCCTGACCCT hTRBV27 hTRBV27 232 ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAG GGAGTCGCC ACCTCTCTGTACT AACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTA CAGCCCCAA TCTGTGCCAGCA TATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGA CCAG GTTTATC TAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCT hTRBV28 hTRBV28 233 ATGGGAATCAGGCTCCTGTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGATGTGAAAGTAACCCAG GGAGTCCGC ACATCTATGTAC AGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAA CAGCACCAA CTCTGTGCCAGC TATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGA CCAG AGTTTATG AAAAGGAGATATTCCTGAGGGGTACAGTGTCTCTAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCT hTRBV29 hTRBV29 234 ATGCTGAGTCTTCTGCTCCTTCTCCTGGGACTAGGCTCTGTGTTCAGTGCTGTCATCTCTCAAAAGCCAAGCAGGG GTGAGCAAC CAGCAGCATATA ATATCTGTCAACGTGGAACCTCCCTGACGATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTCTGGTACC ATGAGCCCT TCTCTGCAGCGT GTCAGCAACCTGGACAGAGCCTGACACTGATCGCAACTGCAAATCAGGGCTCTGAGGCCACATATGAGAGTGGA GAAGA TGAAGA TTTGTCATTGACAAGTTTCCCATCAGCCGCCCAAACCTAACATTCTCAACTCTGACT hTRBV30 hTRBV30 235 ATGCTCTGCTCTCTCCTTGCCCTTCTCCTGGGCACTTTCTTTGGGGTCAGATCTCAGACTATTCATCAATGGCCAG GAGTTCTAA GTGACTCTGGCT CGACCCTGGTGCAGCCTGTGGGCAGCCCGCTCTCTCTGGAGTGCACTGTGGAGGGAACATCAAACCCCAACCTAT GAAGCTCCT TCTATCTCTGTGC ACTGGTACCGACAGGCTGCAGGCAGGGGCCTCCAGCTGCTCTTCTACTCCGTTGGTATTGGCCAGATCAGCTCTG TCTCA CTGGAGTGT AGGTGCCCCAGAATCTCTCAGCCTCCAGACCCCAGGACCGGCAGTTCATCCT 

What is claimed is:
 1. A method for sequencing immune cell receptor genes, comprising (1) providing RNA from immune cells; (2)(a) optionally transcribing the RNA into complementary RNA (cRNA), followed by reverse transcribing the cRNA into complementary DNA (cDNA) using one or more primers that comprise a first adapter sequence, wherein each 5′ end of the cDNA produced by reverse transcription contains the first adapter sequence; (2)(b) if step (2)(a) is not performed, reverse transcribing the RNA into complementary DNA (cDNA), followed by transcribing the cDNA into second strand cDNA using one or more primers that comprise a first adapter sequence, wherein each 5′ end of the cDNA produced by transcribing the cDNA into second strand cDNA contains the first adapter sequence; (3) amplifying the cDNA to produce a first amplification product using a first primer pair comprising a first primer that hybridizes to the first adapter sequence and a second primer that hybridizes to a constant region of immune cell receptor gene; (4) amplifying the first amplification product to produce a second amplification product using a second primer pair, in which i. a first primer of the second primer pair binds to the adapter sequence at the 5′ end of the second amplification product, ii. the second primer of the second primer pair binds to the constant region of immune cell receptor gene in the second amplification product, and iii. the first and second primers comprise adapter sequences for sequencing; and (5) sequencing the second amplification product.
 2. The method according to claim 1, wherein the reverse transcription step results in PCR products ranging from 150-600 bp.
 3. The method according to claim 1 or claim 2, wherein the immune cell receptor genes are T-cell receptor (TCR) genes or B-cell receptor (BCR) genes.
 4. The method of any one of claims 1-3, wherein the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR α chain V segments.
 5. The method of claim 4, wherein the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) comprise one or more of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310.
 6. The method of any one of claims 1-3, wherein the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR β chain V segments.
 7. The method according to claim 6, wherein the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) comprise one or more of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360.
 8. The method of any one of claims 1-3, wherein the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR γ chain V segments.
 9. The method of any one of claims 1-3, wherein the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR δ chain V segments.
 10. The method of any one of claims 1-3, wherein the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to BCR heavy chain V segments.
 11. The method of any one of claims 1-3, wherein the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to BCR light chain V segments.
 12. The method according to any one of claims 1-11, wherein the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) contain a nucleotide barcode sequence.
 13. The method according to claim 12, wherein the nucleotide barcode comprises 6 to 20 nucleotides.
 14. The method according to claim 12 or claim 13, wherein the nucleotide barcode consists of 9 nucleotides.
 15. The method according to any one of claims 12-14, wherein the nucleotide barcode consists of the sequence NNNNTNNNN, NNNNANNNN or HHHHHNNNN.
 16. The method according to any one of claims 1-15, wherein the first adapter sequence of the one or more primers used for the reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) comprises a T7 adapter or an Illumina® adapter.
 17. The method according to any one of claims 1-9 and 12-16, wherein the immune cells are T-cells and wherein the second primer of the first pair of primers hybridizes to the constant region of a TCR gene.
 18. The method according to any one of claims 1-3 and 10-16, wherein the immune cells are B-cells and wherein the second primer of the first pair of primers hybridizes to the constant region of a BCR gene.
 19. The method according to any one of claims 1-18, wherein the sequencing is next generation sequencing.
 20. The method according to any one of claims 1-19, wherein the RNA from the immune cells is obtained by mixing immune cells with carrier cells before RNA extraction.
 21. The method according to any one of claims 1-9 and 12-20, wherein the immune cells are tumor-infiltrating lymphocytes.
 22. The method according to any one of claims 1-9 and 12-20, wherein the immune cells are CD4 or CD8 positive T-cells.
 23. The method according to any one of claims 1-20, wherein the immune cells are purified from peripheral blood mononuclear cells (PBMC) before RNA extraction.
 24. The method according to any one of claims 1-20, wherein the immune cells are part of a mixture of peripheral blood mononuclear cells (PBMC).
 25. The method according to any one of claims 1-24, wherein the immune cells are derived from a mammal.
 26. The method according to claim 25, wherein the mammal is a human or a mouse.
 27. A kit for sequencing of T-cell receptors (TCRs), comprising at least one primer which comprises a TCR α chain V segment portion of any one of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310 and a barcode sequence.
 28. The kit according to claim 27, comprising at least one primer comprising any one of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310.
 29. A kit for sequencing of T-cell receptors (TCRs), comprising at least one primer which comprises a TCR β chain V segment portion of any one of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360 and a barcode sequence.
 30. The kit according to claim 29, comprising at least one primer comprising any one of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360. 